Method and device for the detection of molecular interactions

ABSTRACT

The invention relates to devices and methods for the detection of specific interactions between probe and target molecules. In particular, the invention relates to methods for the qualitative and/or quantitative detection of targets, including: introducing a sample containing targets into a reaction chamber formed between a first surface of the device and a second surface of a device, which is preferably located opposite to the first surface, wherein the distance between the first and the second surface is variable; and detecting the targets.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.14/580,749, filed Dec. 22, 2014, which is a divisional of U.S.application Ser. No. 11/593,021, filed Nov. 6, 2006, which is acontinuation-in-part of, and claims priority under 35 U.S.C. § 120 to,International Patent Application PCT/EP2005/004923, filed on May 6,2005, which designates the United States and claims priority to GermanPatent Application DE 10 2004 022 263, filed May 6, 2004, each of whichis hereby incorporated by reference in its entirety. The presentapplication also claims priority under 35 U.S.C. § 119(a)-(d) to GermanPatent Application DE 10 2005 052 752, filed Nov. 4, 2005, and to GermanPatent Application DE 10 2005 052 713, filed Nov. 4, 2005, each of whichis hereby incorporated by reference in its entirety.

This application also claims priority under 35 U.S.C. § 120 toInternational Patent Application PCT/EP2006/068153, filed Nov. 3, 2006,entitled “Device and Method for the Detection of Particles” and whichdesignates the United States and claims priority to German PatentApplication DE 10 2005 052 752, filed Nov. 4, 2005, each of which isincorporated by reference in its entirety; and to International PatentApplication PCT/EP2006/068155, filed Nov. 3, 2006, entitled “Methods andDevice for the Detection of Molecular Interactions” and which designatesthe United States and claims priority to German Patent Application DE 102005 052 713, filed Nov. 4, 2005, each of which is incorporated byreference in its entirety.

TECHNICAL FIELD

The invention relates to devices and methods for the detection ofspecific interactions between target and probe molecules.

BACKGROUND

Biomedical tests are often based on the detection of an interactionbetween a molecule, which is present in known amount and position (themolecular probe), and an unknown molecule to be detected or unknownmolecules to be detected (the molecular target molecules). Typically,probes are laid out in the form of a substance library on supports, theso-called microarrays or chips, so that a sample can be analyzedsimultaneously at various probes in a parallel manner (see, for example,J. Lockhart, E. A. Winzeler, Genomics, gene expression and DNA arrays;Nature 2000, 405, 827-836). The probes are herein usually immobilized ona suitable matrix, as is for example described in WO 00/12575 (see, forexample, U.S. Pat. No. 5,412,087, WO 98/36827), or syntheticallyproduced (see, for example, U.S. Pat. No. 5,143,854) in a predeterminedmanner for the preparation of the microarrays.

In general, a target molecule labeled with a fluorescence group in theform of a DNA or RNA molecule to a nucleic acid probe of the microarray,that both target molecule and probe molecule are present in the form ofa single-stranded nucleic acid. Efficient and specific hybridization canonly occur between such molecules. Single-stranded nucleic acid targetmolecules and nucleic acid probe molecules can normally be obtained bymeans of heat denaturation and optimal selection of parameters liketemperature, ionic strength, and concentration of helix-destabilizingmolecules. Probes having virtually perfectly complementary, e.g.corresponding to each other, sequences remain paired with the targetsequence (A. A. Leitch, T. Schwarzacher, D. Jackson, I. J. Leitch, 1994,In vitro Hybridisierung, Spektrum Akademischer Verlag,Heidelberg/Berlin/Oxford).

A typical example for the use of microarrays in biological test methodsis the detection of microorganisms in samples in biomedical diagnostics.Herein, it is taken advantage of the fact that the genes for ribosomalRNA (rRNA) are dispersed ubiquitously and have sequence portions, whichare characteristic for the respective species. Thesespecies-characteristic sequences are applied onto a microarray in theform of single-stranded DNA oligonucleotides. The target DNA moleculesto be examined are first isolated from the sample to be examined and areequipped with markers, for example fluorescent markers. Subsequently,the labeled target DNA molecules are incubated in a solution with theprobes fixed on the microarray; nonspecifically occurring interactionsare removed by means of corresponding washing steps and specificinteractions are detected by means of fluorescence-optical evaluation.In this manner, it is possible to detect, for example, severalmicroorganisms simultaneously in one sample by means of one single test.In this test method, the number of detectable microorganismstheoretically only depends on the number of the specific probes, whichhave been applied onto the microarray.

The use of microarrays or probe arrays is not limited to the detectionof target-probe interactions between nucleic acid molecules. Targetscan, for example, also be proteins, which are detected by means ofspecific antibodies functioning as probes. In the same manner,interactions between a protein and low-molecular chemical compounds canbe identified if the protein is immobilized on the array in form of atarget and the chemical compounds, which can, for example, be asubstance library, are immobilized on the array in the form of probes.

Targets can also be analyzed with the aid of conventional immunoassays(for example ELISAs). For instance, antibodies can be immobilized on thebase of a well of a micro well plate. Subsequently, a blood sample to beanalyzed is fed into said well. If the corresponding antigen is presentin the blood sample, it will bind to the immobilized antibody and canthen be detected, for example, via a second antibody bearing afluorescence label.

In the case where cells are to be detected as targets or, for example,the presence of specific antigens on the surface of cells is to beanalyzed, cytometric methods are often employed. Said methods areconventionally based on the fact that corresponding, for examplefluorescence-labeled, antibodies are added to the sample to be analyzed.Said antibodies then bind to the surfaces of the cells or to theantigens presented there. The sample treated in this manner issubsequently guided through a corresponding capillary in a suitabledevice and in a suitable solution. Next to the capillary, a detector isarranged, which detects how often within a specific time interval asignal is triggered by a labeled cell flowing by. The number of cellswanted per volume unit can then, more or less exactly, be determinedfrom the fluctuation rate and the signals counted.

SUMMARY

In one aspect, a method includes: forming a mixture between a firstsurface and a second surface, the mixture including (a) a liquid, (b) afirst complex including a first analyte and a first portion of a firstoptical label, and (c) a second portion of the first optical label, thesecond portion being in an uncomplexed state with respect to the firstanalyte, wherein the second portion has a greater mobility than thefirst complex; reducing a distance between the first surface and thesecond surface and displacing at least a portion of the mixture frombetween the first surface and the second surface without introducing aliquid free of the first optical label between the first surface and thesecond surface; and detecting the first optical label remaining betweenthe first surface and the second surface.

When the complex is immobilized (e.g., linked to a surface), the firstportion of the optical label (i.e., that portion of the optical labelwhich is bound to the analyte in a complex) is similarly immobilized.The second portion of the optical label (i.e., that portion which is notbound to an analyte in a complex) remains free in the liquid and able tomove in or with the liquid. As such, the second portion of the opticallabel has a greater mobility than the first (immobilized) portion.

When the complex is not immobilized (e.g., when the complex includes aparticle, such as a plastic particle or bead, or a cell), the greatermobility of the second portion of the optical label can arise from thedifference in effective sizes of the first portion and second portion ofthe optical label. For example, optical label which is bound to aparticle (i.e., the first portion) can have a greater mass and/orhydrodynamic radius than optical label which is unbound (i.e., thesecond portion). The smaller size of the unbound optical label can allowthe unbound label to move more freely in the liquid, or in other words,to have greater mobility.

The complex can be immobilized with respect to the first surface or thesecond surface. The mixture can further includes (d) a second complexincluding a second analyte differing from the first analyte, and a firstportion of a second optical label, (e) a second portion of the secondoptical label, the second portion being in an uncomplexed state withrespect to the second analyte, wherein the second portion has a greatermobility than the second complex.

The method can further include detecting the second optical labelremaining between the first surface and the second surface. The firstoptical label and the second optical label can be the same. The firstcomplex can be immobilized with respect to the first surface or thesecond surface, and the second complex can be immobilized with respectto the first surface or the second surface, and the first complex isspaced apart from the second complex.

The first complex can include a nucleic acid and the second complex caninclude a nucleic acid. The first complex can be immobilized on thefirst surface. A microarray of nucleic acids can be immobilized on thefirst surface.

The second surface can include a displacement structure. Thedisplacement structure can include an elastic material. The displacementstructure can have a convex shape.

The first complex can include a nucleic acid, a peptide, a protein, anantigen, an antibody, a carbohydrate, a low molecular weight chemicalcompound, or a cell.

The distance between the first surface and the second surface can bereduced so that the mixture is substantially completely displaced frombetween the first surface and the second surface.

The optical label can be a fluorescent label. Detecting the fluorescentlabel can include detection by a fluorescence microscope without anautofocus. Detecting the fluorescent label can include detection by afluorescence microscope including a fixed focus.

The first complex can include a nucleic acid. The method can includeamplifying the nucleic acid between a first surface and a second surfacein a cyclic amplification reaction. The method can include detecting thefirst complex after one or more cycles of the cyclic amplificationreaction.

In another aspect, a device includes a detection zone defined at leastin part between a first surface and a second surface, the detection zonebeing configured to accommodate a mixture including (a) a liquid, (b) afirst complex including a first analyte and a first portion of a firstoptical label, and (c) a second portion of the first optical label, thesecond portion being in an uncomplexed state with respect to the firstanalyte, wherein the second portion has a greater mobility than thefirst complex; an actuator configured to vary a distance between thefirst surface and the second surface and thereby displace at least aportion of the mixture from between the first surface and the secondsurface without introducing a liquid free of the first optical labelbetween the first surface and the second surface; and a detectorconfigured to detect the optical label in the detection zone.

The first analyte can be immobilized on the first surface. The firstanalyte can include a nucleic acid, a peptide, a protein, an antigen, anantibody, a carbohydrate, a low molecular weight chemical compound, or acell. The first analyte can include a nucleic acid.

The device can include a microarray immobilized on the first surface.

The second surface can include a displacement structure. Thedisplacement structure can include an elastic material. The elasticmaterial can be optically transparent and substantially notautofluorescent. The elastic material can be a two-componentplatinum-cross-linking silicone rubber. The elastic material can besilicone oil or a non-cross-linked silicone elastomer. The displacementstructure can have a convex shape.

The actuator can be configured to vary the distance between the firstsurface and the second surface in a range from substantially 0 mm to 1mm. The device can further include a temperature control unit configuredto control the temperature in the detection zone. The temperaturecontrol unit can be integrated into the first surface. The temperaturecontrol unit can include one or more independently controllabletemperature blocks. The temperature blocks can be arranged linearly oron a rotary disk.

The detector can include an optical system. The optical system can be afluorescence-optical system. The fluorescence-optical system can be afluorescence microscope without an autofocus. The optical system can beoperably connected to a spacer which is configured to adjust a spacingbetween a component of the optical system and the second surface. Thesecond surface can be made of a transparent material.

The detection zone can be further defined by compensation zonesconfigured to maintain the volume of the detection zone at asubstantially constant value when the distance between the first surfaceand the second surface is varied.

The first surface can be configured to move relative to the secondsurface. At least a portion of the first surface can be elasticallydeformable. The first surface can include a synthetic elastic material.The actuator can be configured to apply pressure or traction to thefirst surface to thereby vary the distance between the first surface andthe second surface. The actuator can be configured to vibrate the firstsurface.

The second surface can be configured to move relative to the firstsurface. The actuator can be configured to apply pressure or traction tothe second surface to thereby vary the distance between the firstsurface and the second surface.

The distance between the first surface and the second surface can be acapillary gap. The capillary gap can have a thickness in a range ofabout 0 μm to about 100 μm. The device can include a microarrayimmobilized on the first surface. The capillary gap can include at leasttwo sub-chambers, the sub-chambers being in fluid connection with eachother in a first, non-compressed state, and no fluid connection existingbetween the sub-chambers in a second, compressed state. Each sub-chambercan be assigned to a defined zone of said micro-array. At least one ofthe first surface and the second surface can be provided with cavitiesserving as walls between the sub-chambers. The walls between saidsub-chambers can be formed by elastic seals.

The device can include a fluid handling unit configured to purify aprobe solution, reconcentrate a probe solution, control the charging ofthe capillary gap with a fluid, or control the discharge of a fluid fromthe capillary gap. The fluid handling unit and the capillary gap can beconnected to each other via two cannulas, said cannulas being arrangedso that a first cannula ensures the feeding of fluids from the chargingunit and/or reprocessing unit into the capillary gap, and a secondcannula ensures the escape of air from the capillary gap expelled by thesupplied fluids.

The device can include human or machine readable information associatedwith the device, the information describing a substance library,execution of an amplification reaction, or a detection reaction.

The device can include a chamber body made of a electrically conductivematerial. The electrically conductive material can be an electricallyconductive plastic. The electrically conductive plastic can be selectedfrom the group consisting of polyamide with 5-30% carbon fibres,polycarbonate with 5-30% carbon fibres, polyamide with 2-20% stainlesssteel fibres, and PPS with 5-40% carbon fibres.

According to the present invention, methods for the qualitative and/orquantitative detection of molecular interactions between probe andtarget molecules are provided, wherein the replacement and/or theremoval of solutions, i.e. in particular washing or rinsing steps, canbe omitted.

In particular, such methods according to the present invention comprisethe following steps:

-   -   a) introducing a sample containing target molecules into a        reaction chamber having a microarray, said microarray comprising        a substrate onto which probe molecules are immobilized on array        elements; and    -   b) detecting an interaction between the target molecules and the        probe molecules immobilized on the substrate,

wherein after introducing the sample containing target molecules andprior to and during the detection no replacement of solutions in thereaction chamber and/or removal of solutions from the reaction chambertakes place.

Such methods according to the present invention in particular comprisethe following steps: a)

feeding a sample containing targets into a reaction chamber formedbetween a first surface of the device and a second surface of thedevice, which is preferably located opposite said first surface, whereinthe distance between the first and the second surface is variable; b)

detecting the targets.

The method according to the present invention is based on the fact thatsolution components, which are responsible for unspecific backgroundsignals, are displaced from the reaction chamber by means of reducingthe distance between the surfaces. In the case of immobilized probesbound to labeled targets, the number of labeled components of theanalyte solution, which are not bound to probes, is thereby reduced.Herein, it is preferred that no replacement of solutions has to beperformed beforehand.

In one embodiment of the invention, the targets can be detected directlywithout having to fall back upon probes for detection. In otherdetection methods according to the present invention, the targets can bedetected by means of probes, which can, in turn, be immobilized on oneof the surfaces, preferably on a substrate. A further embodimentprovides that the probe molecules are not immobilized, but are presenttogether with the targets in the reaction chamber in a dissolved state.

Furthermore, devices suitable for performing such methods are providedwithin the scope of the present invention.

In particular, within the scope of the present invention a device forthe qualitative and/or quantitative detection of molecular interactionsbetween probe and target molecules is provided, comprising:

-   a) a microarray on a substrate, onto which probe molecules are    immobilized on array elements, said microarray being disposed on a    first surface of the device; and-   b) a reaction chamber formed between the first surface including the    microarray disposed thereon and a second surface,

wherein the distance between the microarray and the second surface isvariable, and wherein the second surface has a displacement structure.

The variability of the distance between the microarray and the secondsurface, which usually represents the detection surface of the inventivedevice, particularly allows for a significant reduction or the completeprevention of a signal background that is caused by labeled targetmolecules having no specific affinity for the probe molecules of themicroarray and thus do not interact with them.

In particular, a device for qualitatively and/or quantitativelydetecting molecular interactions between probe and target molecules isprovided within the scope of the present invention, comprising: areaction chamber formed between a first surface of the device and asecond surface of the device, which is located opposite said firstsurface, wherein the distance between the first and the second surfaceis variable and probe molecules in the reaction chamber are immobilizedon at least one of the two surfaces, preferably on the first surface.

A further device according to the present invention, which is suitablefor performing the methods according to the present invention, comprisesa reaction chamber formed between a first surface of the device and asecond surface of the device, which is located opposite said firstsurface, wherein the distance between the first and the second surfaceis variable and at least one of the two surfaces has a displacementstructure, which is positioned in that region of the surface where thedetection of the targets is supposed to take place. When the twosurfaces approach, the displacement structure leads to a substantiallycomplete displacement of the analyte solution, which is responsible forthe background signal noise, from the region where the detection of thetargets is supposed to take place. Preferably, said displacementstructure is located in the region of the second surface, which islocated opposite that region of the first surface where probe moleculescan be immobilized. Preferably, the displacement structure can be abulge.

In the devices according to the present invention, the plane of thereaction chamber, whereto no probe molecules are immobilized, usually isthe detection plane.

In particular, the variability of the distance between the surfaces ofthe reaction chamber allows the signal background, which is caused bylabeled targets having no specific affinity to the probes employed andtherefore not interacting with the latter, to be considerably reduced orentirely avoided.

In a particular preferred embodiment, the second surface has adisplacement structure located on the surface that is facing themicroarray. This displacement structure causes a substantially completedisplacement of the solution from the reaction chamber if the first andthe second surface approach each other.

According to the present invention, a method for the qualitative and/orquantitative detection of molecular interactions between probe andtarget molecules is also provided, which comprises the following steps:

-   a) introducing a sample comprising target molecules into a reaction    chamber of an inventive device as described above;-   b) detecting an interaction between the target molecules and the    probe molecules immobilized on the substrate.

The methods and devices according to the present invention for thedetection of target molecules are configured in such a way, that as fewinterventions of the practitioner in the reaction chamber as possibleare required for performing the detection method and, optionally, anamplification of the target molecules. This has the essential advantagethat contaminations are avoided. Furthermore, the reproducibility of themethods according to the present invention is considerably increasedcompared to conventional methods, as the inventive method is accessibleto automation due to the minimization of external interventions. Theabove-mentioned advantages play an important role in terms of theapproval of diagnostic methods.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting a schematic view of the device accordingto the present invention comprising a read out device and the processunit.

FIG. 2 is a diagram depicting a view of the process unit according tothe present invention.

FIG. 3 is a diagram depicting an exploded view of the process unitaccording to the present invention comprising detection surface, seal,DNA-chip, and chamber body. The chamber body has a reversibly deformableelastic membrane.

FIG. 4 is a diagram depicting a view of the chamber body having aheating meander embedded by injection-molded synthetic material in theelastic membrane.

FIG. 5 is an illustration of the status of the process unit according tothe present invention in the read out device A) during PCR, B) beforedetection, and C) during detection.

FIG. 6 is an illustration of the mode of function of the process unitaccording to the present invention having membrane seal, compensationfold, and bottom hole. In A), the process unit is shown in homeposition. In B), the process unit is shown in compressed form, in whichthe fluorescent solution between DNA-chip and detection surface isdisplaced.

FIG. 7 is a diagram depicting a view of a rotary disc, whereon fourtemperature blocks are installed. The temperature blocks arethermostatted to one temperature each. By means of rotating the discand/or the process unit, the temperature in the reaction chamber can bealtered.

FIG. 8 is a diagram depicting a view of an exemplary milled and boltedprocess unit.

FIG. 9 is a diagram depicting a view of an exemplary compressing orcrimping device for the process unit according to the present inventionfor the detection of the hybridization signals in a conventionalfluorescence microscope.

FIG. 10 is a diagram depicting a view of a process unit according to thepresent invention having a circuit board as electric connection forheater and temperature sensor. The heater is developed as asemiconductor component.

FIG. 11 is a diagram depicting an exploded view of the process unitshown in FIG. 10.

FIG. 12 is a diagram depicting a view of the straight regression linefor determining the width of a gap filled with fluorophore.

FIG. 13 is a diagram depicting a view of the linearity of thefluorescence signal as the exposure time increases over the meteringrange.

FIG. 14 is a diagram depicting a fluorescence recording of twosuperimposed chips, the gap between which is filled with 200 nM Cy3fluorophore. The intensity of the background is 158 gray values at anexposure time of 0.75 s. The thickness of the gap measured using afluorescence microscope is 40.00 μm. Assuming that the measured grayvalues behave linearly in relation to the exposure time (see FIG. 13),the resulting thickness of the gap, as determined by using equation 1,is 42.6 μm. The values for the thickness of the layer thus obtained arein well agreement with each other.

FIG. 15 is an illustration of the experimental setup for the detectionof DNA arrays without rinsing.

FIG. 16 is a diagram depicting a fluorescence measurement of an arraywith chip pressed against it. The white margins are indicative for thebackground radiation caused by the displaced sample solution.

FIG. 17 is a diagram depicting a decrease of absolute intensities ofsignal and background with reduced thickness of the gap. The differenceof both values is constant throughout the metered region.

FIG. 18 is a diagram depicting a detection of the probe signals bydisplacing background fluorescence. At the left margin, thenon-displaced liquid is shown.

FIG. 19 is a diagram depicting a detection of the probe signals of a DNAarray. The background was corrected by rinsing.

FIG. 20 is a diagram depicting a measurement results of an experimentalcomparison of displacement and replacement of the analyte.

FIG. 21 is a diagram depicting a reference analytics of the PCR in aprocess unit, measured by gel electrophoresis.

FIG. 22 is a diagram depicting a schematic view of a detachable fillingunit for filling reaction cartridges with reactive substances orbuffers. The following reference numbers are used:

-   -   1 Filling unit    -   1.1 Mechanical interface filling unit—cartridge    -   2 Cartridge    -   2.1 Mechanical interface cartridge—filling unit    -   2.2 Seal    -   2.3 Reaction chamber    -   2.4 Preferred opening for the cannulas in the cartridge    -   3 Filling channel    -   3.1 Fluidic and mechanical interface to sample-adding tools    -   3.2 Filling cannula    -   4 Waste channel with waste container    -   4.1 Ventilation hole    -   4.2 Waste cannula

FIG. 23 is a diagram depicting a view of the procedure for filling areaction cartridge by means of a modular filling unit.

FIG. 24 is a diagram depicting a schematic view of an integrated fillingunit for filling reaction cartridges with reactive substances or buffersin the preferred position without penetration of the seal of the chamberbody. The following reference numbers are used:

-   -   1 Filling unit—cartridge    -   1.1 Mechanical interface cartridge—filling unit    -   2 Reaction cartridge    -   2.1 Mechanical interface cartridge—filling unit    -   2.2 Seal    -   2.3 Reaction space    -   2.4 Preferred opening for the cannulas in the cartridge casing    -   3 Filling channel    -   3.1 Fluidic and mechanical interface to sample-introducing tools    -   3.2 Filling cannula    -   4 Waste channel with waste container    -   4.1 Fluidic and mechanical interface to sample-removing units    -   4.2 Waste cannula    -   5 Equipment for preferred position, here: spring

FIG. 25 is a diagram depicting an illustration of the procedure forfilling a reaction cartridge having an integrated filling unit.

FIG. 26 is a diagram depicting a schematic view of an integrated fillingunit having an integrated waste container for filling reactioncartridges with reactive substances or buffers in the preferred positionwithout penetration of the seal of the chamber body. The followingreference numbers are used in addition to the reference numbers in FIG.24:

-   -   4 Waste channel with waste container    -   4.1 Ventilation hole

FIG. 27A is a diagram depicting filling of the reaction space whenremoving the surplus liquid into a waste container or channel; FIG. 27Bis a diagram depicting removal of surplus liquid when reducing thereaction space for detection. The following reference numbers are used:

-   -   1 Reaction chamber    -   2 Seal    -   3 Pressure means    -   4 Fluid interface    -   4.1 Removing cannula    -   4.2 Introducing cannula

FIG. 28 is a diagram depicting a device for processing and detectinginventive reaction cartridges according to example 4. The followingreference numbers are used:

-   -   1 Reaction cartridge    -   1.1 Reaction chamber with microarray    -   1.2 Fluid system interface    -   1.3 Seal of the chamber body    -   1.4 Electric connections for heating system, optionally also        temperature sensors    -   1.5 Chip    -   1.6 Position-securing system for implementing a preferred        position and guiding the cannulas    -   1.7 Cannulas    -   2 Pressure means    -   3 Identification system, for example bar code or data matrix    -   3.1 Identification optics, for example bar code- or data matrix        reader    -   4 Detection optics    -   5 Fluid connections

FIG. 29 is a diagram depicting a reaction cartridge according to Example5.

FIG. 30 is a diagram depicting a recording of the reaction cartridgeaccording to example 5 using a thermal imaging camera at a temperatureof 95° C.

FIG. 31 is a diagram depicting an analysis of the reaction productaccording to example 5 using agarose gel electrophoresis. The referencenumbers indicate:

-   -   1, 5: Positive control from the thermocycler    -   2-4: Reaction products from cartridges    -   6: 100 bp standard

FIG. 32 is a diagram depicting a view exemplarily depicting thearrangement of the displacement structure.

-   -   1: second surface    -   2: first surface    -   3: displacement structure    -   4: solution    -   5: microarray

FIG. 33 is a diagram depicting an analysis of the results of example 6,in which the hybridization results of a probe array during detectionusing the inventive method in a reaction chamber having a displacementstructure are described.

FIG. 34 is a diagram depicting an illustration of devices having areaction chamber with a first and a second surface, whose distance isvariable. (A) depicts probes that are immobilized to one surface of thereaction vessel and the target-containing analyte solution. (B) depictsan arrangement, in which a second surface is provided by a second meanssuch as a tappet. (C) depicts a reaction vessel having elastic surfaceslocated opposite to each other, which represent the first and secondsurface of the reaction chamber.

FIG. 35 is a diagram depicting a illustration of a method according tothe present invention, wherein the probes are not immobilized to a firstsurface of the reaction chamber. Such a method can, for example, beemployed for cytometric applications.

FIG. 36 is a diagram depicting a theoretical illustration of the effectof the chamber and of the thickness of the cells, respectively, on theintensity of the background noise and the signal-to-noise-ratio.

FIG. 37 is a diagram depicting a cytometric determination ofCD4⁺-positive lymphocytes, as described in example 8. (a) depictsunlabeled erythrocytes, (b) depicts CD4⁺-positive lymphocytes.

DETAILED DESCRIPTION

For the description of the present invention, inter alia the followingdefinitions are used.

Within the scope of the present invention, a probe or a probe moleculeor a molecular probe is understood to denote a molecule, which is usedfor detecting other molecules by means of a particular characteristicbinding behavior or a particular reactivity. Each type of molecule,which is suitable for detecting another molecule, i.e. which has aspecific affinity for a specific target, can be used as probe. Here,with respect to the methods according to the present invention, it is noprerequisite that probes can be coupled to solid surfaces, like forexample substrates. In particular, such molecules having a specificaffinity can be used as probes. In a preferred embodiment, these arebiopolymers, in particular biopolymers from the classes of peptides,proteins, antigens, antibodies, carbohydrates, nucleic acids, and/oranalogs thereof and/or copolymers of the above-mentioned biopolymers.Low-molecular chemical compounds, like the typical components ofsubstance libraries, can also be used as probes. In a preferredembodiment, the probes can be coupled to a solid surface.

In particular, nucleic acid molecules having a defined and knownsequence, which are used for the detection of target molecules inhybridization methods, are referred to as probe. Both DNA and RNAmolecules can be used as nucleic acids. For example, the nucleic acidprobes or oligonucleotide probes can be oligonucleotides having a lengthof 10 to 100 bases, preferably of 15 to 50 bases, and particularlypreferably of 20 to 30 bases. Typically, according to the presentinvention, the probes are single-stranded nucleic acid molecules ormolecules of nucleic acid analogs, preferably single-stranded DNAmolecules or RNA molecules having at least one sequence region, which iscomplementary to a sequence region of the target molecules. Depending ondetection method and use, the probes can be immobilized on a solidsupport substrate, e.g. in the form of a microarray. Furthermore,depending on the detection method, they can be labeled radioactively ornon-radioactively, so that they are detectable by means of detectionmethods conventional in the state of the art.

Likewise preferred are antibodies or ligands, which are known tospecifically bind to antigens or cells or cellular structures.

Within the scope of the present invention, a target is generallyunderstood to denote the substance to be detected.

Normally, targets will be molecules; basically, the term “target” isalso supposed to comprise, for example, cellular structures or celltypes, cell classes or cell tissues, however. In some of the methodsaccording to the present invention, for example, the detection ofspecific cells of tissue, like for example fibroblasts, or of the immunesystem, like for example B cells or T cells, is also intended. Dependingon which cellular (sub) structures the probes employed for detecting thetargets are specific for, a detection of cell subsets, like for exampleCD4+ or CD8+ cells can also be performed.

Preferably, within the scope of the present invention, a target or atarget molecule is understood to denote a molecule to be detected bymeans of a molecular probe. In a preferred embodiment of the presentinvention, the targets to be detected are biopolymers from the classesof peptides, proteins, antigens, antibodies, carbohydrates, nucleicacids, and/or analogs thereof and/or mixed polymers of theabove-mentioned biopolymers. Targets can also be low-molecular chemicalcompounds, like components of substance libraries, however. Inorganicmaterials, for example metals, heavy metals, which can be specificallydetected with the aid of detection reagents (for example complex formingagents), are also understood as targets.

If the targets according to the present invention are nucleic acids ornucleic acid molecules, which are detected by means of a hybridizationagainst probes located on a probe array, said target molecules normallyinclude sequences of a length of 40 to 10,000 bases, preferably of 60 to2,000 bases, also preferably of 60 to 1,000 bases, particularlypreferably of 60 to 500 bases and most preferably of 60 to 150 bases.Optionally, their sequence includes the sequences of primers as well asthe sequence regions of the template, which are defined by the primers.In particular, the target molecules can be single-stranded ordouble-stranded nucleic acid molecules, one or both strands of which arelabeled radioactively or non-radioactively, so that they are detectableby means of a detection method conventional in the state of the art.

According to the present invention, a target sequence denotes thesequence region of the target, which is detected by means ofhybridization with the probe. According to the present invention, thisis also referred to as said region being addressed by the probe.

Within the scope of the present invention, a substance library isunderstood to denote a multiplicity of different molecules, preferablyat least two to 1,000,000 different molecules, particularly preferablyat least 10 to 10,000 different molecules, and most preferably between100 to 1,000 different molecules. In special embodiments, a substancelibrary can also comprise only at least 50 or less or at least 30,000different molecules.

Within the scope of the present invention, a support element or supportor substrate is understood to denote a component whereon probes can beimmobilized. For the case that no probes are immobilized, the termsubstrate is supposed to define that region of the first surface or thesecond surface where the detection of the targets is supposed to takeplace in the state of the compressed reaction chamber. In both cases(i.e. immobilized probe or non-immobilized probe) the substrate does nothave to be an individual component, but it can be a region of the firstsurface (immobilized probe) or of the first and/or second surface(non-immobilized probe). Substrates can be, for example, object supportsor wafers, but also ceramic materials, however. In a special embodiment,the probes can also be immobilized directly on one of the preferablyopposite surfaces of the reaction chamber, preferably on a partition ofthe surface.

Within the scope of the present invention, a probe array is understoodto denote an array of molecular probes or a substance library on asupport, wherein the position of each probe is determined separately.Preferably, the array comprises defined sites or predetermined regions,so-called array elements, which are particularly preferably arranged ina specific pattern, wherein each array element typically comprises onlyone species of probes. The arrangement of the molecules or probes on thesupport can be generated by means of covalent or non-covalentinteractions. Therefore, the probes are arranged on the side of thesupport that is facing the reaction chamber. A position within thearrangement, i.e. within the array, is usually referred to as a spot.

Within the scope of the present invention, an array element or apredetermined region or a spot or an array spot is understood to denotean area on a surface, which is determined for the deposition of amolecular probe, the entirety of all occupied array elements being theprobe array.

Within the scope of the present invention, a support element or asupport or a substance library support or a substrate can denote a solidbody, on which a probe array is located. The support, which is usuallydenoted a substrate or a matrix, can be, for example, an object slide ora wafer or ceramic materials. In a specific embodiment, the probes mayalso be immobilized on the first surface, preferably in a portion of thefirst surface.

Of course, the person skilled in the art is aware of the fact that withmicroarrays the immobilization of the probes also takes place onsubstrates, i.e. in defined spatial layout of the spots. The entirety ofmolecules laid out on the substrate in array layout or the substancelibrary laid out in array layout on the substrate or on the detectionplane is often also referred to as chip, microarray, DNA chip, probearray, etc., which are used synonymously for the purposes of the presentinvention.

Within the scope of the present invention, a detection surface (plane)is understood to denote the second surface of the inventive device.Preferably, during detection the probes deposited on the microarray aresubstantially located in the detection plane, in particular due to thefact that the distance between microarray and second surface is reducedto about zero.

For example, the detection plane is preferably understood to denote thesurface of the device according to the present invention, whereon noprobes are immobilized when the detection is performed, for example, inincident light. If the detection is performed in transmitted light,however, no detection surface has to be defined, or, in this case, thefirst surface and the second surface can function as detection plane.Preferably, the probes laid out on the other surface are substantiallylocated in the plane of the second surface during the detection of theinteraction between probes and targets, in particular due to the factthat the distance between the probes of the first surface and the secondsurface is reduced to about zero.

A displacement structure is denoted to be a structure, preferablyconfigured as a bulge of the second surface, that is located, at leastpartially, in the area of the second surface, which is located oppositeto the microarray and arranged on the side of the second surface that isfacing the microarray.

The displacer has the function to substantially displace the(fluorescent) solution between the detection surface (second surface)and the first surface. In the embodiments described below without havinga displacement structure, there can occasionally arise the problem thatremnants of fluorescent solution remain between the two surfaces, thuscausing the background noise mentioned above. This can be avoided byemploying a displacement structure, which is preferably configured as abulge of the second surface.

Numerous materials can be used for displacement structures, with elasticor soft and ductile materials being preferred. Preferably, saidmaterials are optically transparent and not autofluorescent,respectively, so that they do not adversely interfere with detection.Suitable materials may be, for example, silicone rubbers or siliconeelastomers, “classic” rubbers, polyurethanes, acrylics, acrylates, andTPE. Particularly preferred are two-component platinum-cross-linkingsilicone rubbers, such as PDMS. These are optically transparent, notautofluorescent and biologically inert.

Likewise, a liquid, which cannot be mixed with the fluorescent analytesolution or cannot be dissolved in the fluorescent analyte solution, forexample silicone oil, may be employed. For example, a silicone rubber(such as Dow Corning Sylgard 184); which does not necessarily need to becured, may be employed. These materials are preferably notautofluorescent.

The softer the material employed, the better it compensates forunevenness in the surface and for the potential roughness of a surfaceof the microarray, respectively. If liquid materials are used, theyshould preferably wet the surface in order to allow an optimaldisplacement of the fluorescent solution.

The displacement structure, which is preferably configured as bulge,should have an outer shape, which allows for displacing liquid from thereaction chamber and/or from the surface of the microarray in an asefficient manner as possible. Thus, geometric shapes having a convexsurface are preferred. As a matter of course, planar, rectangular, orround shapes may be employed as well. In the compressed state, thedisplacement structure may, for example, cover the entire microarray oronly parts thereof. Convex displacement structures are preferred, sincethey only contact one point of the opposite surface when the reactionchamber is compressed, and, as compression continues, ensure that theopposite surface becomes substantially completely covered, wherein theliquid is laterally displaced from the reaction chamber or from thesurface of the microarray. Geometric shapes achieving similar goals arelikewise preferred.

The displacement structure may be glued on, dripped on, or deposited andfixed by a suitable means. Other methods, however, are not excluded. Forinstance, the displacement structure may be configured as one elementtogether with the second surface and thus be manufactured from onepiece.

The principle of a displacement structure is exemplarily depicted inFIG. 32.

In performing the methods according to the present invention, whereinthe detection of the targets is performed either without probes or bymeans of non-immobilized probes, both surfaces can likewise be used asdetection plane. In this case, the detection of the targets or of thetarget probes is preferably also performed during the detection in thedetection plane, which in turn is preferably achieved by the distancebetween the first surface and second surface being reduced to aboutzero. For the case of employing immobilized probes, the substancesupport usually is part of the chamber body, wherein the substancesupport can be made of a different material than the rest of the chamberbody.

Within the scope of the present invention, a chamber body is understoodto denote the solid body forming the reaction chamber. Usually, thesubstance library support or the chip is part of the chamber body,wherein the substance library support can be made of a differentmaterial than the rest of the chamber body.

Within the scope of the present invention, a reaction chamber or areaction space is understood to denote the space formed between thefirst surface and the second surface and preferably designed in form ofa variable capillary gap. Preferably, the reaction space can belaterally limited by side walls, which can, for example, be implementedas elastic seals. In the case of immobilized probes, these are locatedon the side facing the interior of the reaction chamber.

Within the scope of the present invention, a reaction chamber or areaction space is understood to denote the space formed betweenmicroarray and second surface or detection surface and preferablyconfigured in form of a variable capillary gap. The reaction space islaterally limited by side walls, which can, for example, be implementedas elastic seals. The probes immobilized on the microarray are locatedon the side facing the interior of the reaction chamber.

The base area of the reaction chamber or of the reaction space isdefined by the first surface or the second surface of the array. Thedistance between second surface or detection surface and the surface ofthe substrate or of the microarray is referred to as thickness of thereaction space or of the reaction chamber or of the capillary gap,respectively. The base of the reaction chamber or of the reaction spaceis defined by the size of the first surface or the second surface. Thebase of the reaction chamber can also be defined by sections of thefirst or the second surface. In particular, the distance between firstand second surface, and preferably between second surface and surface ofthe immobilized probes on the first surface is referred to as thicknessof the reaction space or of the reaction chamber or of the capillarygap. Within the scope of the present invention, a reaction space usuallyhas a reaction space usually has a thickness providing a volume optimalfor the corresponding interaction to be detected. The reaction space canhave, for example, a thickness of at most 1 cm, preferably of at most 5mm, more preferably of at most 3 mm and most preferably of at most 1 mm.

Within the scope of the present invention, the distance between themicroarray and the second surface is understood to denote the distancebetween the surface of the microarray substrate, i.e. of the side of themicroarray facing the reaction space, and the side of the second surfacefacing the reaction space. If the distance between microarray and secondsurface is about zero, this means that the surface of the substraterests evenly on the second surface.

Within the scope of the present invention, a capillary gap is understoodto denote a reaction space, which can be filled by means of capillaryforces acting between the microarray and the second surface. Usually, acapillary gap has a small thickness, for example of at most 1 mm,preferably of at most 750 μm, and particularly preferably of at most 500μm. Furthermore, according to the present invention, a thickness of thecapillary gap in the range of 10 μm to 300 μm, of 15 μm to 200 μm or of25 μm to 150 μm is preferred. In special embodiments of the presentinvention, the capillary gap has a thickness of 50 μm, 60 μm, 70 μm, 80μm or 90 μm. Within the scope of the present invention, the reactionspace or reaction chamber will not be referred to as a capillary gapanymore, if the reaction space or the reaction chamber has a thicknessof more than 2 mm.

Within the scope of the present invention, a cartridge or reactioncartridge is understood to denote a unit consisting of the reactionchamber with a chamber body and a corresponding casing.

The devices employable for the methods according to the presentinvention can come in different forms. They can be, for example,reaction vessels like cuvettes or other reaction vessels, provided theyhave two opposite surfaces, whose distance from each other is variable.An example is to be found in FIG. 5 or FIG. 6. Thus, all reactionvessels having elastic surfaces, which allow the formation of acapillary gap of variable size, can be used as devices for the methodsaccording to the present invention. In particular, the first or thesecond surface of the reaction vessels should be elastic in thoseregions where probes are immobilized.

The first and second surface of the devices employable for the methodsaccording to the present invention do not necessarily have to becomponents of the same object. For instance, a reaction chamber can begenerated by means of, for example, inserting an object like a tappetinto a reaction vessel like a cuvette. Said object, which is insertedinto the reaction vessel, should be dimensioned in such a way that itcan be snug-fit into the reaction vessel while leaving a gaprepresenting the reaction chamber but being narrow enough to effectdisplacement of the solution responsible for the signal noise. In suchembodiments, for example, the first surface of the reaction chamber canbe provided by the reaction vessel and the second surface can beprovided by the object to be inserted.

In all these embodiments, either the first and/or preferably the secondsurface is made of a transparent material, which allows opticaldetection of the targets.

Within the scope of the present invention, a confocal fluorescencedetection system is understood to denote a fluorescence detectionsystem, wherein the object is illuminated in the focal plane of theobjective by means of a point light source. Herein, point light source,object and point light detector are located on exactly opticallyconjugated planes. Examples for confocal systems are described in A.Diaspro, Confocal and 2-photon-microscopy: Foundations, Applications andAdvances, Wiley-Liss, 2002.

Within the scope of the present invention, a fluorescence optical systemimaging the entire volume of the reaction chamber is understood todenote a non-confocal fluorescence detection system, i.e. a fluorescencedetection system, wherein the illumination by means of a point lightsource is not limited to the object. Such a fluorescence detectionsystem therefore has no focal limitation.

Conventional arrays or microarrays within the scope of the presentinvention comprise about 50 to 10,000, preferably 150 to 2,000 differentspecies of probe molecules on a, preferably square, surface of 1 mm to 4mm×1 mm to 4 mm, preferably of 2 mm×2 mm, for example. In furtherembodiments within the scope of the present invention, microarrayscomprise about 50 to about 80,000, preferably about 100 to about 65,000,particularly preferably about 1,000 to about 10,000 different species ofprobe molecules on a surface of several mm² to several cm², preferablyabout 1 mm² to 10 cm², particularly preferably 2 mm² to 1 cm², and mostpreferably about 4 mm² to 6.25 mm². For example, a conventionalmicroarray has 100 to 65,000 different species of probe molecules on asurface of 2 mm×2 mm.

Within the scope of the present invention, a label or a marker isunderstood to denote a detectable unit, for example a fluorophore or ananchor group, to which a detectable unit can be coupled.

Within the scope of the present invention, a duplication oramplification reaction comprises typically 10 to 50 or moreamplification cycles, preferably about 25 to 45 cycles, particularlypreferably about 40 cycles. Within the scope of the present invention, acyclic amplification reaction is preferably a polymerase chain reaction(PCR).

Within the scope of the present invention, an amplification productdenotes a product resulting from the duplication or the copying or theamplification of the nucleic acid molecules to be amplified by means ofthe cyclic amplification reaction, preferably by means of the PCR. Anucleic acid molecule amplified by means of PCR is also referred to asPCR product.

Within the scope of the present invention, the denaturation temperatureis understood to denote the temperature at which double-stranded DNA isseparated in the amplification cycle. Usually, the denaturationtemperature, in particular in a PCR, is higher than 90° C., preferablyabout 95° C.

Within the scope of the present invention, the annealing temperature isunderstood to denote the temperature at which the primers hybridize tothe nucleic acid to be detected. Usually, the annealing temperature, inparticular in a PCR, lies in a range of 50° C. to 65° C. and preferablyis about 60° C.

Within the scope of the present invention, the chain extensiontemperature or extension temperature is understood to denote thetemperature at which the nucleic acid is synthesized by means ofinsertion of the monomer components. Usually, the extension temperature,in particular in a PCR, lies within a range of about 68° C. to about 75°C. and preferably is about 72° C.

Within the scope of the present invention, an oligonucleotide primer orprimer denotes an oligonucleotide, which binds or hybridizes the DNA tobe detected, also referred to as target DNA, wherein the synthesis ofthe complementary strand of the DNA to be detected in a cyclicamplification reaction starts from the binding site. In particular,primer denotes a short DNA or RNA oligonucleotide having preferablyabout 12 to 30 bases, which is complementary to a portion of a largerDNA or RNA molecule and has a free 3-OH group at its 3′-end. Due to saidfree 3′OH group, the primer can serve as substrate for any optional DNAor RNA polymerases, which synthesize nucleotides to the primer in5′-3′-direction. Herein, the sequence of the newly synthesizednucleotides is predetermined by that sequence of the template hybridizedwith the primer, which lies beyond the free 3′OH group of the primer.Primers of conventional length comprise between 12 and 50 nucleotides,preferably between 15 and 30 nucleotides.

A double-stranded nucleic acid molecule or a nucleic acid strand servingas template for the synthesis of complementary nucleic acid strands isusually referred to as template or template strand.

Within the scope of the present invention, a molecular interaction or aninteraction is understood to denote a specific, covalent or non-covalentbond between a target molecule and an immobilized probe molecule. In apreferred embodiment of the present invention, the interaction betweenprobe and target molecules is a hybridization.

The formation of double-stranded nucleic acid molecules or duplexmolecules from complementary single-stranded nucleic acid molecules isreferred to as hybridization. Herein, the association preferably alwaysoccurs in pairs of A and T or G and C. Within the scope of ahybridization, for example DNA-DNA duplexes, DNA-RNA duplexes, orRNA-RNA duplexes can be formed. By means of a hybridization, duplexeswith nucleic acid analogs can also be formed, like for example DNA-PNAduplexes, RNA-PNA duplexes, DNA-LNA duplexes, and RNA-LNA duplexes.Hybridization experiments are usually used for detecting the sequencecomplementarity and therefore the identity of two different nucleic acidmolecules.

Within the scope of the present invention, processing is understood todenote purification, concentration, labeling, amplification,interaction, hybridization, and/or washing and rinsing steps as well asfurther method steps performed when detecting targets by using substancelibraries. Detection itself does not fall under the term processing.

Within the scope of the present invention, a sample or sample solutionor analyte or solution is a liquid to be analyzed, which in particularcontains the target molecules to be detected and, optionally, to beamplified. Furthermore, beside conventional additives such as buffers,such a solution may inter alia also contain substances required forperforming amplification reactions, like primers.

Within the scope of the present invention, a replacement of solutions inthe reaction chamber from the reaction chamber refers, in particular, torinsing or washing steps. The replacement of solutions serves, forexample, for removing molecules labeled with detectable markers, whichdo not specifically interact with probes on the microarray, by replacingthe sample solution with a non-labeled solution after the interactionhas occurred. Molecules not specifically interacting with probes on themicroarray are, for example, primers labeled with a detectable marker,which have not been converted during the amplification reaction, ortarget molecules labeled with a detectable marker, which do not have acomplementary probe on the array, which specifically interacts with saidtarget molecule.

Within the scope of the present invention, a removal of solutions fromthe reaction chamber is understood to denote steps, by means of whichmolecules labeled with detectable markers, which do not specificallyinteract with probes, are removed from the reaction chamber. Moleculesnot specifically interacting with probes are, for example, primerslabeled with a detectable marker, which have not been converted duringthe amplification reaction, or target molecules labeled with adetectable marker, which do not have a complementary probe on the array,which specifically interacts with said target molecule.

If, within the scope of the present invention, no replacement ofsolutions in the reaction chamber and/or removal of solutions from thereaction chamber is performed between feeding the sample containingtarget molecules into a reaction chamber and detecting the interaction,it is, however, conceivable that during this time period solutions canadditionally be introduced into the reaction chamber without performinga replacement or removal of the solutions already present in thereaction chamber.

An object of the present invention thus comprises a method for thequalitative and/or quantitative detection of targets and, in particularof molecular interactions between probe and target molecules, inparticular comprising the following steps:

a) introducing a sample containing target molecules into a reactionchamber, which is formed between a first surface of a device and asecond surface of a device, wherein the distance between the first andthe second surface is variable;

b) detecting the targets.

Preferably, no replacement of solutions in the reaction chamber and/orremoval of solutions from the reaction chamber has to be performed afterfeeding the sample containing target molecules and before or during thedetection.

Such a method can, for example, be employed for absorption measurementsin solutions without requiring probes for detecting the targets.According to Lambert-Beer's Law, the extinction of a solution isproportional to the concentration of the solution and the layerthickness in which the solution is present. Conventionally, withsolutions having too high a concentration, i.e. with solutions having anabsorption so high that it cannot be determined reliably anymore, adilution series is prepared, which is subsequently analyzed. Thepossibility shown herein is based on the variation of the secondparameter, the layer thickness. Alternatively to reducing theconcentration, the layer thickness is herein reduced until theabsorption of the solution can be measured reliably. As the thickness ofthe measured layer can be determined, the concentration of the solutioncan be determined reliably.

Such an embodiment of the method according to the present invention can,for example, be used for determining the concentrations of solutions.

A further object of the present invention comprises a method for thequalitative and/or quantitative detection of molecular interactionsbetween probe and target molecules, in particular comprising thefollowing steps:

a) introducing a sample containing target molecules into a reactionchamber formed between a first surface of a device and a second surfaceof a device, wherein the distance between the first and the secondsurface is variable;

b) detecting an interaction between the target molecules and the probemolecules immobilized on the substrate.

In this aspect of the present invention, it is a substantialcharacteristic of the method according to the present invention that thedetection of an interaction between the target molecules to be detectedand the probe molecules immobilized on the substrate of the microarrayis performed without replacing solutions in the reaction chamber orremoving solutions from the reaction chamber. That is, detecting theinteraction between targets and probes can be performed without rinsingor washing steps being required subsequently to the interaction reactionand/or without removing molecules, which do not specifically interactwith probes on the microarray, from the reaction chamber subsequently tothe interaction reaction.

In this embodiment of the method according to the present invention, theprobes can, but do not have to, be immobilized on the first surface.This is to be explained in general with reference to FIG. 34.

For instance, a probe can be immobilized at the first surface of any oneof the devices mentioned, wherein the probes are not arranged in arraylayout. It is thus a method for detecting targets by means of employingprobe molecules bound to a surface, characterized in that

-   -   the surface of the first surface is coated completely or        partially with at least one type of probe molecules;    -   the detection is performed in a step, wherein all reagents        required are mixed and no further rinsing steps are required for        removing material not bound to probes;    -   the detection is preferably performed under chemical balance        conditions;    -   and the material not bound to probes is removed from the        detection volume, for example, by means of mechanical spatial        displacement. This can be achieved, for example, by means of        compressing the reaction chamber or by means of inserting a        separate displacement body (for example a piston) or a liquid        insoluble in the actual reaction medium, like for example a        mineral oil in aqueous reaction media.

In FIG. 34, A) represents a cuvette with a reaction solution containingtarget molecules. The inside of the cuvette is coated with probemolecules, which can, for example, be antibodies. In said cuvette, thesolution to be examined including the detection reagents is incubatedunder suitable conditions. Herein, binding of the targets to the probestakes place.

In FIG. 34, B), the cuvette is then shown with targets bound to probes,wherein the superfluous reaction solution is displaced by means of asnug-fit tappet, which preferably forms a small gap as reaction chamber.The tappet can consist of an optically transparent material, for examplequartz glass, so that a detection of the bound targets can be performedthrough the tappet. The tappet can, however, also consist of othermaterials, for example of a non-fluorescent optically opaque material.Detection could then, for example, be performed by means of incidentlight (e.g., fluorescence microscopy) detection. In addition, the tappetcan be coated with a material improving the displacing effect at surfacecontact. Such materials referred to as “displacers” will be described indetail in the following. In both cases, at least one surface of thecuvette preferably also consists of transparent materials.

The embodiment shown in FIG. 34, B) represents an example, wherein thefirst and the second surface of the reaction chamber are provided bydifferent objects, namely tappet and cuvette.

In FIG. 34, C), the cuvette is again shown with bound targets, whereinthis time the superfluous reaction solution is displaced from thedetection volume by means of compressing the opposite surfaces of thecuvette. In this case, the cuvette again consists of transparent and,moreover, elastic materials. Detection can, for example, take placealong the axis of a device for compressing.

This embodiment of the method according to the present invention can,for example, be employed in a sample carousel. Herein, several sampletubules are arranged, for example, in a high throughput screeningmachine in a carousel. The machine pipettes the samples to be analyzedinto the tubules. Said tubules are then guided past a detector andpressed against said detector in a defined manner by means of a suitabledevice.

In both embodiments described, the solution containing non-bound butlabeled targets is reduced in the detection region. This leads toreduction of the unspecific background signal and to improvement of thesignal-to-noise ratio.

Of course, the person skilled in the art is aware of the fact that thedescribed embodiments of the methods according to the present inventioncan also be performed in reaction vessels other than the cuvettedescribed. Among those are, inter alia, the reaction cartridgesdescribed in the following.

In principle, the reaction chamber can adopt any exterior shape; it mustbe possible, however, to reduce its volume.

All hitherto described embodiments as well as all the other embodimentsof the present invention have the special advantage that it is possibleto analyze the increase of a product formed during the reaction by meansof binding the formed product as target to the probes immobilized at thefirst surface of the reaction chamber, subsequently compressing thereaction chamber in order to reduce its volume, and detecting thesignal. Subsequently, the reaction chamber can be relaxed again so thatthe reaction can continue. After a suitable time period, the reactionchamber is again compressed and the increase of the signal is detectedby means of the increased amount of product bound to the probes. Thisprocedure can be performed as often as is desired.

This method is also suitable for creating binding kinetics by means ofcoating the reaction chamber, for example, with a protein as probe andsubsequently adding a further protein, whose binding to the firstprotein is to be examined, as target. Said second protein can, forexample, be directly defined with a fluorescence marker or it can belabeled with an antibody labeled with a fluorescence marker. In relationto the time elapsed, the increase of the signal in the reaction chamberis then detected, wherein the solution covering the probe is reversiblydisplaced by means of the above-described reduction of the chambervolume.

The method according to the present invention, i.e. the detection oftarget/probe interactions by means of reducing the volume of thereaction chamber in order to effect the removal of solutions responsiblefor unspecific signals, can also be employed without immobilization ofthe probes being required.

This principle shall be explained by way of employing the methodsaccording to the present invention in cytometric methods.

Normally, cytometric methods are based on specially labeled cells beingguided through a corresponding capillary in a suitable solution. Next tothe capillary, a detector is arranged, which detects how often within aspecific time interval a signal is triggered by a labeled cell flowingby. The number of cells wanted per volume unit can then, more or lessexactly, be determined from the fluctuation rate and the signalscounted.

In the method according to the present invention, non-immobilized probesare employed. Said probes are combined with the targets to be detected.In cytometric applications, the targets can, for example, be cell types.The probes can, for example, be cell-type-specific fluorescence-labeledantibodies.

The solution containing probes and targets is fed into the reactionchamber, the detection volume of which is variable. The reaction chamberis then compressed in a defined manner so that the signal to bedetected, which is coming form the surfaces of the cells, rises abovethe signal intensity of the environment.

FIG. 35 illustrates this principle. In FIG. 35, A), the targets to bedetected are placed in a reaction chamber, the detection volume of whichis variable in the manner described above, together with suitabledetection reagents and are incubated under suitable conditions. Herein,the probes (for example antibodies recognizing specific antigens on thecell surface) bind at the cell surface of the targets, which leads to arelative increase in density of probe molecules compared to thesurrounding solution. In FIG. 35, B), the reaction chamber is compressedto form a defined gap for detection, wherein analyte solution is pressedout of the reaction chamber, whose volume can be determined. A specificportion of labeled cells remains in the capillary gap, whosefluorescence can then be detected as they fluoresce brighter than theremaining fluorescent components of the analyte solution. According toFIG. 35, C), the labeled cells can then be imaged in front of thebackground and be counted, for example, by means of a suitable software.By repeating the steps B) and C) once or several times, a mean value ofthe number of cells detected per volume unit can be generated and thusthe exactness of the measurement can be further increased.

FIG. 36 illustrates the theoretical background of the detection oftargets like cells or particles in front of a fluorescent backgroundbased on the enrichment of fluorescence-labeled molecules on the surfaceof the particles. The calculation is based on the assumption that aprobe molecule occupies an area of 2,500 nm² (yielding a density ofprobe molecules of 400 molecules per μm² cell surface) or 400 nm² (whichrather corresponds to the real conditions, yielding a maximum occupationdensity of 2,500 molecules per μm² cell surface). For calculating thebackground fluorescence, quenching effects and the like are omitted. Itis assumed that background fluorescence behaves proportionally to thenumber of fluorescent molecules in the corresponding volume element.

It can clearly be seen that a distinction from the background is alreadypossible with a low occupation density of the cell surface withfluorescent material, if the detection volume is correspondinglynarrowed.

In principle, different targets can be detected in a parallel mannerwith this method. To this end, different probes (for example differentlyspecific antibodies bearing different fluorescence dyes) can be employedon the one hand; on the other hand, however, the geometric shape of thetargets to be detected can also be analyzed with correspondingmagnification. Here, for example, beads of different sizes or differentgeometries, which specifically bind in different ways, can be employed.

Basically, the methods according to the present invention have in commonthat they are performed in reaction chambers, which are formed by afirst surface and a second surface, wherein the second surface islocated opposite said first surface and the distance between thesurfaces is variable in such a way that the volume of the reactionchamber can be reduced to capillary gap size and smaller.

The reaction chambers described in the following for reaction cartridgescan in particular be employed as devices in such applications.

An object of the present invention thus comprises a method for thequalitative and/or quantitative detection of targets and, in particular,of molecular interactions between probe and target molecules, inparticular comprising the following steps:

a) introducing a sample containing target molecules into a reactionchamber having a microarray, said microarray comprising a substrate ontowhich probe molecules are immobilized on array elements; and

b) detecting an interaction between the target molecules and the probemolecules immobilized on the substrate,

wherein after introducing the sample containing target molecules andprior to and during the detection no replacement of solutions in thereaction chamber and/or removal of solutions from the reaction chambertakes place.

In some embodiments, detection of an interaction between the targetmolecules to be detected and the probe molecules immobilized on themicroarray substrate occurs without a replacement of solutions in thereaction chamber or the removal of solutions from the reaction chamber.That is, the detection of the interaction between targets and probes canoccur without the requirement of rinsing or washing steps after theinteraction reaction has taken place and/or without the removal ofmolecules from the reaction chamber that do not specifically interactwith probes on the microarray after the interaction reaction has takenplace

This may particularly be ensured in the inventive method by means offoci-selective detection methods, such as confocal techniques or theevanescent de-coupling of excitation light (TIRF) in the samplesubstrate based on the use of a depth-selective illumination due to, forexample, total reflection, or the use of methods based on waveguides.Such foci-selective methods are to be particularly preferred in caseswhen a further exclusion of the background signals caused by thefluorescence molecules present in the liquid, i.e. not hybridized, inorder to increase sensitivity. By using fluorescence-labeled targetmolecules, the specific interaction signals can thus be discriminatedfrom the background fluorescence by employing methods such as totalinternal reflection fluorescence microscopy (TIRF) or confocalfluorescence microscopy.

Examples for this are CCD-based detectors, which implement theexcitation of the fluorophores in the dark field by means of incidentlight or transmitted light for the purpose of discriminating opticaleffects like dispersion and reflections (see for example C. E. Hooper etal., Quantitative Photon Imaging in the Life Sciences Using IntensifiedCCD Cameras, Journal of Bioluminescence and Chemoluminescence (1990),337-344). Further alternatives for fluorescence detection systems, whichcan be used in the method according to the present invention, are whitelight setups, like for example described in WO 00/12759, WO 00/25113,and WO 96/27025; confocal systems, like for example described in U.S.Pat. Nos. 5,324,633, 6,027,880, 5,585,639, and WO 00/12759; confocalexcitation systems based on Nipkow discs in confocal imaging, as forexample described in U.S. Pat. No. 5,760,950; systems based onstructured excitation distribution, as for example described in WO98/57151; large-scale integrated fluorescence detection systems usingmicro-optics, like for example described in WO 99/27140; and laserscanning systems, as for example described in WO 00/12759. A generalprocedure of fluorescence detection methods using such conventionalfluorescence detection systems is, for example, described in U.S. Pat.No. 5,324,633.

The devices described in WO 2004/087951, wherein the reaction chamber isformed by a capillary gap, are particularly suitable for performing adetection method according to the present invention without replacingsolutions in the reaction chamber and/or removing solutions from thereaction chamber. The relevant contents of WO 2004/087951 are herebyexplicitly referred to.

In a further embodiment of this aspect of the present invention,replacing and/or removing solutions from the reaction chamber is avoidedby performing the detection by means of detecting the mass alteration onthe array surface, as described, for example, in WO 03/004699. Therelevant contents of WO 03/004699 are hereby explicitly referred to.

In a further embodiment of this aspect of the present invention,replacing and/or removing solutions from the reaction chamber is avoidedby performing the detection by means of detecting acoustic surfacewaves, as is described, for example, in Z. Guttenberg et al., Lab Chip.2005; 5(3):308-17.

In a further embodiment of this aspect of the present invention,replacing and/or removing solutions from the reaction chamber is avoidedby performing the detection by means of electrochemical detection viaelectrodes on the surface of the substrate onto which the probes areimmobilized, like, for example, by means of measuring the alteration ofredox potentials (see, for example, X. Zhu et al., Lab Chip. 2004;4(6):581-7) or cyclic voltametry (see, for example, J. Liu et al., AnalChem. 2005; 77(9):2756-2761; J. Wang, Anal Chem. 2003; 75(15):3941-5).

In a further embodiment of this aspect of the present invention,replacing and/or removing solutions from the reaction chamber is avoidedby performing the detection by means of electric detection viaelectrodes on the surface of the substrate, onto which the probes areimmobilized, like, for example, by means of impedance measurement (see,inter alia, S. M. Radke et al., Biosens Bioelectron. 2005;20(8):1662-7).

In a further embodiment of this aspect of the present invention,replacing and/or removing solutions from the reaction chamber is avoidedby employing a substrate having FRET probes (FRET, fluorescenceresonance energy transfer). The use of such FRET probes is based on theformation of fluorescence quencher pairs, so that a fluorescence signalonly occurs, if a target molecule has bound to the complementary probeon the surface. The use of FRET probes is, for example, described in B.Liu et al., PNAS 2005, 102, 3, 589-593; K. Usui et al., Mol Divers.2004; 8(3):209-18; J. A. Cruz-Aguado et al., Anal Chem. 2004;76(14):4182-8 and J. Szollosi et al., J Biotechnol. 2002; 82(3):251-66.

In a further particularly preferred embodiment of this aspect of thepresent invention, replacing and/or removing solutions from the reactionchamber is avoided by means of employing an inventive device for thequalitative and/or quantitative detection of molecular interactionsbetween probe and target molecules, as detailed below, wherein thedevice comprises:

a) a microarray on a substrate, onto which probe molecules areimmobilized on array elements, said microarray being disposed on a firstsurface of the device; and

b) a reaction chamber formed between the first surface including themicroarray disposed thereon and a second surface,

wherein the distance between the microarray and the second surface isvariable.

In a further particularly preferred embodiment of this aspect of thepresent invention, replacing and/or removing solutions from the reactionchamber is avoided by means of employing a device according to thepresent invention for qualitatively and/or quantitatively detectingmolecular interactions between probe and target molecules, as isdescribed in detail in the following, wherein the device comprises areaction chamber formed between a first surface of the device and asecond surface of the device, wherein the distance between the first andthe second surface is variable and probe molecules are immobilized inthe reaction chamber on at least one of the two surfaces.

In an equally preferred embodiment of this aspect of the presentinvention, replacing and/or removing solutions from the reaction chamberis avoided by means of employing a device according to the presentinvention for qualitatively and/or quantitatively detecting molecularinteractions between probe and target molecules, as is described indetail in the following, wherein the device comprises a reaction chamberformed between a first surface of the device and a second surface of thedevice, wherein the distance between the first and the second surface isvariable and one of the two surfaces has a displacement structure, whichleads to volume reduction in a region of the reaction chamber when thetwo surfaces approach. Preferably, said displacement structure islocated in that region of the second surface, which is located oppositethe region of the first surface where probe molecules are immobilized.

A further object of the present invention relates to the use of FRETprobe molecules, as described above, and/or detection methods selectedfrom the group consisting of total internal reflection fluorescencemicroscopy (TIRF), as described above, confocal fluorescence microscopy,as described above, methods for detecting mass alterations, as describedabove, methods for detecting acoustic surface waves, as described above,methods for the electrochemical and/or electric detection, as describedabove, for avoiding replacement of solutions in a reaction chamberand/or removal of solutions from a reaction chamber during or afterintroducing a sample containing target molecules into the reactionchamber and before or during the detection in a method for thequalitative and/or quantitative detection of molecular interactionsbetween probe and target molecules, in particular comprising thefollowing steps:

-   -   a) introducing a sample containing target molecules into a        reaction chamber of one of the devices described above;    -   b) detecting the targets, in particular by detecting an        interaction between the target molecules, preferably immobilized        on a substrate on one of the two surfaces.

A further object of the present invention particularly relates to adevice for the qualitative and/or quantitative detection of molecularinteractions between probe ans target molecules, comprising:

a) a microarray on a substrate, onto which probe molecules areimmobilized on array elements, said microarray being disposed on a firstsurface of the device; and

b) a reaction chamber formed between the first surface including themicroarray disposed thereon and a second surface,

wherein the distance between the microarray and the second surface isvariable.

After the interaction between probe molecules and target molecules hastaken place, an undesired background is caused by the labeled moleculespresent in the sample solution, which do not interact with the probemolecules. In case the probe and/or target molecules are nucleic acidsand/or nucleic acid analogs, said background is caused, in particular,by the labeled primers and/or labeled nucleic acids present in thesample solution, which are not hybridized with the probe molecules.

A known possibility of removing disturbing background signals is thereplacement of the sample solution after completed interaction with anon-labeled, for example non-fluorescent, solution. However, thisvariant is generally lavish and prone to interference owing tocorrosion, aging of the solutions and impermeability problems.

In some embodiments, the volume of that region of the reaction chamberwhere the detection of the targets takes place can be reduced by meansof varying the distance between the first and the second surface. Avariable distance between first and second surface means that thereaction chamber of the device according to the present invention iscompressible. In particular, the distance between first and secondsurface is variable in such a way that the first surface can rest evenlyand/or reversibly on the second surface or can be pressed onto thelatter. In one embodiment, the distance can, in particular, be reducedin that region of the first or second surface where the probes areimmobilized and/or where the displacement structure is mounted.

A compressible reaction chamber therefore allows displacement of samplesolution containing labeled molecules, which do not interact with theprobe molecules and therefore constitute an undesired background, byreducing the distance between the microarray and the second surfacebefore performing the detection. In this manner, a detection ofinteractions between probe and target molecules using any opticaldetection systems is possible without replacing the sample solution witha non-labeled solution before the detection. For example, simplefluorescence-microscopic imaging of the DNA chip for detecting theinteraction signals by means of the device according to the presentinvention without replacing the sample solution with a non-labeled, inparticular weakly fluorescent, liquid, is possible. In particular, thisapplies, if the inventive device has a displacement structure located onthe second surface, as described above

It is finally ensured, in particular by means of the embodiments of thedevice according to the present invention described in the following,that focusing of optical detection systems is not necessary anymore.Thus, the device according to the present invention allows, for example,the use of a simple fluorescence microscope device without autofocusfunction as reading device for the detection of the hybridizationbetween targets and probes without necessitating liquid-handling stepslike, in particular, washing steps, for removing target molecules notbound to the array, like for example non-hybridized target nucleicacids, contrarily to the fluorescence-optical detection systems hithertoused for the detection of nucleic acids. This also applies, if theinventive device has a displacement structure located on the secondsurface, as described above.

Despite multifunctional sample treatment and analysis, which is feasibleby means of the device according to the present invention, a verycost-efficient system for detecting and, optionally, amplifying targetmolecules in a sample is provided. The devices according to the presentinvention, in particular in connection with an optical detection system,are furthermore robust to such an extent that they are also suitable formobile use.

By means of suitably selecting the substrates with or withoutimmobilized targets, processing protocols, and analysis chemicals, thedevice according to the present invention can be employed for the mostdifferent types of gene analyses, like for example predispositiondiagnostics, germ diagnostics and typing. Thus, a complete geneticanalysis is conductible with little equipment effort in the deviceaccording to the present invention, which can also be implemented as adisposable cartridge. Therefore, the device according to the presentinvention allows performing detection methods on-site, for exampleduring blood donation. A measured result can be quickly obtained,preferably within 0.5 to 2 hours. All the steps practicable with thedevice according to the present invention, like purification,processing, amplification of nucleic acids, and the actual hybridizationcan be conducted automatically. The operator only needs to be familiarwith sample withdrawal, sample feeding into the device according to thepresent invention, and taking notice of the analysis results.

The same applies for concentration determinations of targets withoutemploying probes, if the targets have absorbing properties or forcytometric applications or target/probe interactions, which are, forexample, based on protein/protein interactions.

Preferably, the distance between the first surface (e.g., microarray)and the second surface is variable in a range of about 0 to about 1 mm.Further preferred lower limits for the distance between microarray andsecond surface are about 0.1 μm, about 1 μm, and about 10 μm. Furtherpreferred upper limits for the distance between the microarray andsecond surface are about 0.01 mm, about 0.5 mm, about 1 mm and mostpreferably about 0.3 mm. Surprisingly, the interaction between probesand targets is not even affected if the distance between substratesurface and second surface is approximately zero or about zero. Thisalso applies, if the inventive device has a displacement structurelocated on the second surface, as described above.

Preferably, the device according to the present invention furthercomprises a detection system. Herein, it is preferred that the detectionsystem is an optical system. Examples for systems suitable within thescope of the present invention are detection systems based onfluorescence, optical absorption, resonance transfer, and the like.Preferably, the optical detection system is a fluorescence-opticalsystem. Particularly preferably, the fluorescence-optical system is afluorescence microscope without autofocus, for example a fluorescencemicroscope with fixed focus.

In a further embodiment, the detection system is connected with at leastone spacer, which adjusts a distance between the detection system andthe second surface when resting upon the second surface. If the distancebetween the first and the second surface is about zero, the spacer alsodetermines the distance between the surface of the chip and the opticalsystem of the detection device. It is thus possible to keep the varianceof the distance between optical detection device and microarray surfacevery small. The variance only comprises the thickness variance of thesecond surface, in general a glass surface, the deflection of the secondsurface, and the thickness of a layer caused by possible impurities atthe pressing surfaces between chip and detection plane or between spacerand detection plane. This renders re-focusing for bringing the opticalsystem into focus unnecessary, which considerably simplifies theoperation of the device and/or renders an expensive autofocusinstallation unnecessary.

In a further embodiment, laterally limiting compensation zones, whichkeep the volume in the reaction chamber basically constant when thedistance between microarray and second surface is reduced, are providedfor the reaction space formed between the first and the second surface.This also applies, if the inventive device has a displacement structurelocated on the second surface, as described above.

In addition, the reaction space formed between the first and the secondsurface is preferably laterally limited by elastic seals. Particularlypreferably, the elastic seals are made of silicone rubber. This alsoapplies, if the inventive device has a displacement structure located onthe second surface, as described above.

In order to ensure the detection of interactions between probe andtarget molecules, the second surface is preferably made of an opticallytransparent material, particularly preferably glass. The same alsoapplies to an optional displacement structure which, however, may alsobe made of an elastic and optionally transparent material.

In a further embodiment of the device according to the presentinvention, the first surface is, at least in the region of themicroarray configured in such a way that the first surface can be guidedrelatively to the second surface in such a way that the distance betweenthe microarray and the second surface is variable.

Herein, the first surface can, at least in the region on which theprobes can be immobilized, be configured in such a way that this regioncan be guided in the direction towards the second surface so that thedistance between the first surface and the second surface can be reducedand/or that the microarray can be guided in a direction away from thesecond surface in a way that that the distance between the microarrayand the second surface can be increased. This also applies, if theinventive device has a displacement structure located on the secondsurface, as described above.

In this embodiment, it is preferred that the first surface can, at leastin the region of the microarray, be elastically deformed. Particularlypreferably, the first surface is made of an elastic synthetic material,for example an elastic membrane. This also applies, if the inventivedevice has a displacement structure located on the second surface, asdescribed above.

It can further be preferred that the first surface is formed by twosuperimposed layers, wherein an outer layer of the two superimposedlayers has a cut-out at least in the region below the microarray. Inthis embodiment, it is preferred that an inner layer of the twosuperimposed layers is formed by an elastic seal or a sealing membrane,which usually also limits the reaction space laterally (see FIG. 6). Thesealing membrane can be guided toward the second surface. The sealingmembrane closes a recess in the outer layer, which usually correspondsto the lower side of the chamber body. During the performance of a PCRin the reaction chamber, an internal pressure, which renders thereaction chamber pressure-resistant despite the relatively labilesealing membrane, is generated due to the higher temperatures prevailingin a PCR. This embodiment thus corresponds to a self-closing valve. Inorder to ensure the elasticity of the sealing membrane, the membrane ispreferably provided with a compensation fold (see FIG. 6). This alsoapplies, if the inventive device has a displacement structure located onthe second surface, as described above.

It can further be provided that the device comprises at least one means,by which the microarray or probes can be guided relatively to the secondsurface. In the following, said means will be referred to as means forguiding the first surface. Said means for guiding the first surface ispreferably selected from the group consisting of a rod, a pin, a tappet,and a screw. This also applies, if the inventive device has adisplacement structure located on the second surface, as describedabove.

Herein, the device can comprise at least one means for guiding the firstsurface, by which the first surface can be guided towards the secondsurface in such a way that the distance between the first and the secondsurface can be reduced and/or by which the first surface can be guidedaway from the second surface in such a way that the distance between thefirst surface and the second surface can be increased. This alsoapplies, if the inventive device has a displacement structure located onthe second surface, as described above.

Particularly preferably, at least one region of the first surface can beguided relatively to the second surface by applying pressure and/ortraction, which is exerted on the first surface by the means.

Herein, the above-mentioned spacers resting on the second surface canserve as holders for the means for guiding the first surface.

It can further be preferred that the first surface can be caused tovibrate by the means for guiding the first surface, in particular tovibrate at a frequency of 10 to 30 Hz, particularly preferably of about20 Hz. In this manner, bubbles present above the chip, which wouldimpede a detection, can be removed and/or the interaction speed, forexample the hybridization speed, can be increased by a thorough mixingowing to the vibration of the means for guiding the first surface.

It may also be preferred that the second surface can be guidedrelatively to the first surface in such a way that the distance betweenthe first and the second surface is variable.

There, the second surface can be guided relatively to the first surfacein such a way that the distance between the first and the second surfacecan be reduced and/or that the distance between the first and the secondsurface can be increased.

In particular, this can be ensured by the second surface being guidablerelatively to the first surface by means of the spacer exerting pressureand/or traction on the second surface, such that the distance betweenthe first and the second surface is variable at least in that area wherethe detection of the target is to be performed.

In a further preferred embodiment of the device according to the presentinvention, both the first surface and the second surface can be guidedin such a way that the distance between the first and the second surfaceis variable.

Preferably, the first and/or second surface consist of elastic materialsat least in those regions where they can be guided relatively to oneanother. Preferably, such materials are silicone elastomers, like forexample Sylgard 184, rubber, silicone rubber or also elastic syntheticmaterials. TEP, polyurethanes and acrylics or acrylates can also be usedas materials.

In a further preferred embodiment, said elastic materials are opticallytransparent, like for example silicone rubber (for example Sylgard 184).

Preferably, the materials mentioned above are not fluorescent.

Particularly preferred are two-component platinum-cross-linking siliconerubbers (like for example PDMS (Sylgard 184 by Dow Corning)). These aretransparent, biologically inert, and not fluorescent.

In a further embodiment, the device according to the present inventionis developed in such a way that, already in the original state, themicroarray mounted on the first surface rests, preferably evenly, on thesecond surface forming the detection plane. The first surface can beguided in such a way that the distance between the microarray and thesecond surface can be increased. Herein, the first surface is preferablymade of an elastic material.

In a further embodiment of the device according to the presentinvention, the first surface is developed in a pivotable manner around arotation axis. The rotation axis divides the first surface into twosides. In this embodiment, the microarray is arranged on a firstflanking portion of the first surface. Preferably, the rotation axis forthe swiveling motion runs through the center of the first surface, i.e.the two flanking portions preferably are of equal size. The firstsurface is preferably made of an elastic material.

In a first position of the pivotable first surface, the first surface isarranged basically parallel to the second surface. In the firstposition, the surface of the microarray contacts the second surfacebasically evenly, i.e. the substrate surface with the probe moleculesimmobilized thereon is basically not moistened by the sample solution.In said first position, a space, which is also referred to as processingchamber in the following, is formed between the second flanking portionof the first surface and the second surface. Said processing chamber canserve as chamber for processing the sample solution.

In a second position of the pivotable first surface, the first surfaceis arranged at an angle other than 1800 in relation to the secondsurface. In said second position, the surface of the microarray does notcontact the second surface, i.e. the probe molecules immobilized on thesubstrate of the microarray are freely accessible for the targetmolecules present in the sample solution and can therefore interact withthe latter. In the second position, the processing chamber iscompressed.

The pivotable first surface can preferably be swiveled by means ofexerting traction on the first flanking portion of the first surfaceand/or by means of exerting pressure on the second flanking portion ofthe first surface. Pressure and/or traction can be exerted by means of ameans for guiding the first surface, as described above.

The preceding embodiments optionally have a displacement structurelocated on the second surface, as described above, namely on the side ofthe second surface that is facing the microarray. Thereby, thedisplacement structure is positioned in such a way that it is locatedopposite to the microarray and evenly rests on it in the compressedstate, wherein during compression the analyte solution being issubstantially displaced from the reaction chamber and the surface of themicroarray, respectively.

The chip or the substrate or the first surface onto which probes can beimmobilized can preferably consist of silicon, ceramic materials likealuminum oxide ceramics, borofloat glasses, quartz glass, single-crystalCaF₂, sapphire discs, topaz, PMMA, polycarbonate, and/or polystyrene.The selection of the materials is also to be made dependent on theintended use of the device or the chip. If, for example, the chip isused for characterizing PCR products, only those materials may be used,which can resist a temperature of 95° C.

Preferably, the chips are functionalized by means of nucleic acidmolecules, in particular by means of DNA or RNA molecules. However, theycan also be functionalized by means of peptides and/or proteins, likefor example antibodies, receptor molecules, pharmaceutically activepeptides, and/or hormones, carbohydrates and/or mixed polymers of saidbiopolymers.

In a further preferred embodiment, the molecular probes are immobilizedon the substrate surface via a polymeric linker, for example a modifiedsilane layer. Such a polymeric linker can serve for the derivativepreparation of the substrate surface and therefore for theimmobilization of the molecular probes. In the case of covalent bindingof the probes, polymers, for example silanes, are used, which have beenfunctionalized or modified by means of reactive functionalities likeepoxides or aldehydes. Furthermore, the person skilled in the art isalso familiar with the activation of a surface by means ofisothiocyanate, succinimide, and imido esters. To this end,amino-functionalized surfaces are often correspondingly derivatized.Furthermore, the addition of coupling reagents, like for exampledicyclohexylcarbodiimide, can ensure corresponding immobilizations ofthe molecular probes

The chamber body of the reaction chamber preferably consists ofmaterials like glass, synthetic material, and/or metals like high-gradesteel, aluminum, and brass. For its manufacturing, for example syntheticmaterials suitable for injection molding can be used. Inter alia,synthetic materials like macrolon, nylon, PMMA, and teflon areconceivable. In special embodiments, electrically conductive syntheticmaterials like polyamide with 5 to 30% carbon fibers, polycarbonate with5 to 30% carbon fibers, polyamide with 2 to 20% stainless steel fibers,and PPS with 5 to 40% carbon fibers and, in particular, 20 to 30% carbonfibers are preferred. Alternatively and/or in addition, the reactionspace between first and second surface can be closed by means of septa,which, for example, allow filling of the reaction space by means ofsyringes. In a preferred embodiment, the chamber body consists ofoptically transparent materials like glass, PMMA, polycarbonate,polystyrene, and/or topaz. Herein, the selection of materials is to beadjusted to the intended use of the device. For example, thetemperatures the device will be exposed to are to be considered whenselecting the materials. If, for example, the device is to be used forperforming a PCR, for example, only those synthetic materials may beused, which remain stable for longer periods at temperatures like 95° C.

In particular, the chamber body is developed in such a way that themicroarray can be pressed against the second surface evenly and/orreversibly with its active side, i.e. the side of the array, whereon thenucleic acid probes are immobilized.

In a special embodiment, the device according to the present inventioncomprises modules selected from the group consisting of a chamber body,preferably made of a synthetic material, a septum or a seal sealing thereaction chamber, a DNA chip, and/or a second optically transparentsurface, preferably a glass pane, wherein the second surface canoptionally also serve as chip simultaneously (see FIG. 2 and FIG. 3). Inthis embodiment, chamber body and seal are developed elastically, sothat the DNA chip can be pressed evenly and reversibly to the glasscover with its active side. Thereby, the labeled analysis liquid locatedbetween DNA chip and detection surface is entirely displaced (see FIG. 5and FIG. 6). In this manner, a highly sensitive fluorescence detection,for example a computer-imaging fluorescence microscopy, can be conductedwithout being impaired by a background fluorescence of the samplesolution.

Preferably, the second surface of the chamber body consists oftransparent materials like glass and/or optically permeable syntheticmaterials, for example PMNA, polycarbonate, polystyrene, or acryl. Thedisplacement structures mentioned above, if present, may be made ofthese materials or of the above-mentioned materials.

Preferably, the reaction chamber is developed between the second surfaceand the microarray in the form of a capillary gap having variablethickness. By forming a capillary gap between chip and detection plane,capillary forces can be utilized for safely filling the reactionchamber. Said capillary forces already occur in the non-compressed stateof the reaction chamber; they can, however, be increased by compressingthe reaction chamber. Particularly preferably, the capillary gap has athickness in the range of about 0 μm to about 100 μm. This also applies,if a displacement structure is present, as described above

From the possibility of being able to compress the reaction space andtherefore to reduce the width of the gap between microarray anddetection plane, further possibilities of handling the liquid within thereaction chamber arise. Thus, in a further embodiment of the presentinvention, several sub-chambers are provided instead of one singlechamber, wherein the partitions between said sub-chambers do not reachthe height of the second surface, so that a fluid connection isgenerated between the sub-chambers in a non-compressed state of thereaction chamber. By compressing the reaction chamber, the chambers canbe separated. Thus, by compressing, the partitions between the chamberscan be operated like valves.

A special embodiment of said sub-chambers separated by valves is thesubdivision of the reaction space of the device according to the presentinvention into different PCR chambers. In each chamber, individualprimers are presented. In the beginning, the sub-chambers aresimultaneously filled with the analyte. Subsequently, the reaction spaceis compressed. Afterwards, the reaction space is subjected to thetemperature cycle for the PCR. As each sub-chamber is filled withdifferent primers, a different amplification reaction takes place ineach chamber. An exchange between the chambers does not occur.

After the PCR has been performed, hybridization takes place. Herein,each sub-chamber can contain an individual chip region or an individualchip. However, it is also possible to facilitate a fluid connectionbetween the sub-chambers by increasing the distance between microarrayand second surface, so that the different substances to be amplified mixand in this manner hybridize to a chip surface.

The advantage of this embodiment having sub-chambers separated by valvesis the increase in multiplexity of the PCR, i.e. the number ofindependent PCRs with one sample, which is limited for biochemicalreasons in a one-stage reaction. Thus, it is possible to adjust thenumber of PCRs to the possible number of probes on the chip surface.

In a further embodiment of the present invention, the reaction chamberthus comprises at least two sub-chambers, wherein in a firstnon-compressed state the sub-chambers are in fluid connection and in asecond compressed state there is no fluid connection between thesub-chambers.

Particularly preferably, each sub-chamber is assigned to a definedregion of the microarray.

In particular, the sub-chambers can be formed by equipping themicroarray and/or the second surface with cavities, which serve as wallsbetween the sub-chambers.

Particularly preferably, the walls between the sub-chambers are formedby elastic seals.

Of course, this embodiment of the process unit having sub-chambersseparated by valves can arbitrarily be combined with any of theabove-described compression principles.

In a further embodiment of the device according to the presentinvention, the first surface is made of a partially deformable elasticmaterial, for example an elastic membrane. In that only a part of thereaction space can be compressed, sub-chambers, wherein the chip isguided toward the second surface, sub-chambers, which cannot beseparated from each other, and sub-chambers, which cannot be altered,can, inter alia, be generated. Thereby, simple pump systems, which can,for example, be used for pumping salts into the hybridization chamber atthe end of an amplification reaction, can be implemented in the reactionspace. This can, for example, be advantageous for optimizing thechemical hybridization conditions of the PCR buffer, wherein the PCRbuffer is optimized only for the conduction of the PCR.

When subdividing the reaction chamber into several sub-chambers, it ispreferred to use several means for agitating. Usually, the means foragitating are identical with the means for guiding the first surface.Thereby, individual chambers can be specifically agitated. This can, forexample, be appropriate for implementing separate amplification spacesand/or hybridization spaces.

Of course, this embodiment of the device according to the presentinvention having several means for agitating can also be arbitrarilycombined with any of the above-described compression principles.

The above-described components or modules of the device according to thepresent invention selected from the group consisting of a chamber body,seals laterally limiting the reaction space, micro-array, and detectionplane form the so-called process unit of the inventive device. In theprocess unit, PCR, hybridization reactions, detection and/or evaluationcan be performed. This is similarly true for probes and targets being,for example, antibodies and proteins to be detected, thus a PCR does nothave to be carried out. However, the following explanations areparticularly done with regard to detection techniques using nucleic acidtargets and -probes. However, it is clear to the person skilled in theart how to modify the units described in the following to adapt them forother applications.

Preferably, the process unit of the device according to the presentinvention is constructed in a modular manner. This means that theprocess unit can comprise any arbitrary combination of the modules. Themodules can also be exchanged during analysis.

The preceding embodiments optionally have a displacement structurelocated on the second surface, as described above, namely on the side ofthe second surface that is facing the microarray. Thereby, thedisplacement structure is positioned in such a way that it is locatedopposite to the microarray and evenly rests on it in the compressedstate, wherein during compression the analyte solution being issubstantially displaced from the reaction chamber and the surface of themicroarray, respectively.

In a further preferred embodiment, the device according to the presentinvention in addition comprises a temperature controlling and/orregulating unit for controlling and/or regulating the temperature in thereaction chamber. Such a temperature controlling and/or regulating unitfor controlling and/or regulating the temperature in the reactionchamber in particular comprises heating and/or cooling elements ortemperature blocks. Herein, the heating and/or cooling elements or thetemperature blocks can be arranged in such a way that they contact thefirst surface and/or the second surface. By means of contacting both thefirst and the second surface, particularly efficient temperaturecontrolling and regulating is ensured.

In this embodiment, the substrate of the microarray or the first surfaceand/or the second surface is connected with heating and/or coolingelements and/or temperature blocks and should then preferably consist ofmaterials with good heat-conducting properties. Such heat conductivematerials offer the considerable advantage of ensuring a homogenoustemperature profile throughout the entire surface of the reaction spaceand therefore allowing temperature-dependent reactions, like for examplea PCR, to be conducted homogenously throughout the entire reactionchamber, delivering high yields, and controllably or regulatably withhigh accuracy.

Thus, in a preferred embodiment, the substrate of the microarray or thefirst surface or the second surface consist of materials having a goodheat conductivity, preferably having a heat conductivity in a range of15 to 500 Wm⁻¹K⁻¹, particularly preferably in a range of 50 to 300Wm⁻¹K⁻¹, and most preferably in a range of 100 to 200 Wm⁻¹K⁻¹, whereinthe materials are usually not optically transparent. Examples forsuitable heat conductive materials are silicon, ceramic materials likealuminum oxide ceramics, and/or metals like high-grade steel, aluminum,copper, or brass.

If the substrate of the microarray or the first surface or the secondsurface of the device according to the present invention substantiallyconsists of ceramic materials, the use of aluminum oxide ceramics ispreferred. Examples for such aluminum oxide ceramics are the ceramicsA-473, A-476, and A-493 by Kyocera (Neuss, Germany).

Preferably, the substrate of the microarray or the first surface or thesecond surface is equipped with optionally miniaturized temperaturesensors and/or electrodes or has heater structures on its back side,i.e. the side facing away from the reaction chamber, so that temperingthe sample liquid and mixing the sample liquid by means of an inducedelectro-osmotic flow is possible.

The temperature sensors, for example, can be developed asnickel-chromium thin film resistance temperature sensors.

The electrodes, for example, can be developed as gold-titaniumelectrodes and, in particular, as quadrupole.

The heating and/or cooling elements can preferably be selected in such away that fast heating and cooling of the liquid in the reaction chamberis possible. Herein, fast heating and cooling is understood to denotethat temperature alterations in a range of 0.2 K/s to 30 K/s, preferablyof 0.5 K/s to 15 K/s, particularly preferably of 2 K/s to 15 K/s, andmost preferably of 8 K/s to 12 K/s or about 10 K/s can be mediated bythe heating and/or cooling elements. Preferably, temperature alterationsof 1 K/s to 10 K/s can also be mediated by the heating and/or coolingelements.

The heating and/or cooling elements, for example resistance heaters,can, for example, be developed as nickel-chromium thin film resistanceheaters.

For further details on the specification and dimension of thetemperature sensors, heating and/or cooling elements or means forincreasing the temperature and of the electrodes, it is referred to thecontents of the International Patent Application WO 01/02094.

In a preferred embodiment, tempering of the reaction chamber is ensuredby using a chamber body consisting of electrically conductive material.Such an electrically conductive material is preferably an electricallyconductive synthetic material, like for example polyamide, optionallyhaving 5 to 30% carbon fibers, polycarbonate, optionally having 5 to 30%carbon fibers, and/or polyamide, optionally having 2 to 20% stainlesssteel fibers. Preferably, PPS (polyphenylenesulfide) with 5 to 40%carbon fibers, particularly preferably 20 to 30% carbon fibers, is usedas electrically conductive synthetic material. It is further preferredthat the chamber body is developed in such a way that it has swellingsand tapers. Such swellings or tapers in the chamber body allow specificheating of the reaction chamber or the corresponding surfaces.Furthermore, the use of such volume conductors has the advantage that,also with optionally lower heat conductivity of the material used,homogenous tempering of the chamber or the corresponding surfaces isensured, as heat is released in each volume element.

Coupling and educing heat into the reaction space can be conducted indifferent ways. Inter alia, it is intended to bring in heat via externalmicrowave radiation, internal or external resistance heating, internalinduction coils or surfaces, water cooling and heating, friction,irradiation with light, in particular with IR light, air cooling and/orheating, friction, temperature emitters, and peltier elements.

Measuring the temperature in the reaction space can be conducted indifferent ways, for example by means of integrated resistance sensors,semi-conductor sensors, light waveguide sensors, polychromatic dyes,polychromatic liquid crystals, external pyrometers like IR radiationand/or temperature sensors of all types, which are integrated in themeans for guiding the microarray.

Measuring the temperature in the reaction chamber can furthermore beconducted by means of integrating a temperature sensor in the chamberbody, for example by means of injection in the course of the productionprocess of the chamber body, by means of non-contact measurement withthe aid of a pyrometer, an IR sensor, and/or thermopiles, by means ofcontact measurement, for example with a thermal sensor integrated in thedevice and contacting a suitable surface or a suitable volume of thechamber body or the chamber, by means of measuring thetemperature-dependent alteration of the refraction index at thedetection plane, by means of measuring the temperature-dependentalteration of the color of specific molecules, for example in thesolution, on the probe array, or in the chamber seal, and/or by means ofmeasuring the temperature-dependent alteration of the pH-value of thesolution used by means of measuring the color alteration of apH-sensitive indicator, for example by means of measuring itsabsorption.

Furthermore, automatic limitation of temperature can occur due to asurge of the resistance of the heater, wherein the correspondingthreshold temperature preferably lies in a range of 95° C. to 110° C.When reaching the threshold temperature, the resistance of the heatersurges, whereby virtually no current flows and therefore virtually noheat is emitted anymore. In particular, polymers, like electricallyconductive polyamides, whose resistance increases at the thresholdtemperature due to the alteration of the matrix of the polymer or aphase alteration, can be used for such heaters.

In one embodiment, the temperature controlling and regulating unit canbe integrated in the first surface and/or the second surface. In saidembodiment, the process unit is, in particular, equipped with a heater(see FIG. 4), which serves for implementing the temperature alterationsin PCR and hybridization.

Preferably, the process unit has a low heat capacity, so that maximumtemperature alteration speeds of, for example, at least 5 K/s arepracticable at a low power demand. In order to ensure fast cooling ofthe process unit, another preferred embodiment intends providing acooling system, for example an air cooling system.

Preferably, cooling of the process unit can also be achieved by means ofpermanently tempering the space surrounding the process unit to alowered temperature and thereby passively cooling the cartridge. Thisrenders active cooling of the reaction cartridge unnecessary.

In a further embodiment, the temperature controlling and regulating unitcan comprise temperature blocks, which are each pre-heated to a definedtemperature. In said embodiment the process unit, in particular, has nointegrated heater. Owing to the omission of an integrated heatingsystem, the process unit can be provided even more cost-efficiently.

Heat transfer between the temperature blocks of the temperaturecontrolling and regulating unit is preferably ensured in that thetemperature blocks contact the first surface and/or second surface ofthe device according to the present invention. Preferably, thetemperature blocks can be arranged linearly or on a rotary disc and, forexample, be integrated in the detection device in this manner. FIG. 7shows a rotary disc having several temperature blocks, each of which isadjusted to a defined temperature. By means of exchanging thetemperature blocks below the process unit, the process unit is broughtto a specific temperature defined by the temperature block. Preferably,the temperature blocks are manufactured in such a way, that they have asignificantly higher heat capacity than the process unit, so thatmaximal temperature alteration speeds of, for example, at least 5 K/sare also practicable in this embodiment. Preferably, the temperatureblocks are only thermostaticized instead of heated or cooled, so thatthe energy demand is also minimal in this case. In this embodiment,cooling or heating the process unit can be omitted.

In a further embodiment, the temperature controlling and regulating unitis integrated in the means for guiding the first surface and/or in themeans for agitating, and/or in the spacer. In this embodiment, heattransfer is conducted by means of contacting the means and/or the spacerwith the first surface and/or the second surface.

Preferably, the device additionally comprises a reprocessing unit forpurifying and/or re-concentrating the sample solution and/or forcontrolling the loading and unloading of the reaction chamber withfluids. Within the scope of the present invention, fluids are understoodto denote liquids and gases. Furthermore, the analysis solution can bere-buffered in the reprocessing unit. The reprocessing unit can finallyalso be used for providing the necessary analysis chemicals. Theconnection of the fluid containers with the reaction chamber can, forexample, be developed as described in the International PatentApplication WO 01/02094.

In this embodiment, the reaction chamber and the reprocessing unit areparticularly preferably connected via two cannulas, wherein the cannulasare arranged in such a way that a first cannula ensures the feeding offluids from the reprocessing unit into the reaction chamber and a secondcannula ensures the escape of air dislocated by the fed fluids from thereaction chamber. A sample fed into the reprocessing unit can thus reachthe reaction chamber of the process unit via the cannulas. To this end,the cannulas are arranged in such a way that they reach into thereaction chamber via the cannula guide.

The reprocessing unit can be developed in such a way that it can beseparated from the process unit. After filling the reaction chamber withthe sample solution and, optionally, with further reaction liquids, thereprocessing unit can thus be separated from the process unit,preferably be disengaged, and, optionally, be discarded.

All preceding embodiments optionally have a displacement structurelocated on the second surface, as described above, namely on the side ofthe second surface that is facing the microarray. Thereby, thedisplacement structure is positioned in such a way that it is locatedopposite to the microarray and evenly rests on it in the compressedstate, wherein during compression the analyte solution being issubstantially displaced from the reaction chamber and the surface of themicroarray, respectively.

In the following, embodiments of integrated or non-integrated units forfilling the reaction chamber, which will also be referred to in thefollowing as filling unit or reprocessing unit, will be described. Theseembodiments may have the above-mentioned displacements structureslocated on the second surface.

Conventionally, the reaction solution is brought into a specific openingof the filling unit by means of a suitable tool, for example, a pipette.The transport of liquids into the device is performed via the pressureexerted by the pipette or by means of another pressure-generating tool,like for example a syringe or an automated unit, which is, for example,a functional component of a processing automat.

Preferably, the filling unit is developed for manual operation in anergonomically suitable way. Furthermore, it preferably has easilyaccessible additional openings at the outsides for feeding the reactivesubstances.

Preferably, a filling unit furthermore has a suitable fluid interfacefor penetrating the seal of the chamber body. To this end, specificcannulas are used, which, for example, consist of high-grade steel orpolymers and usually have a diameter of 0.05 mm to 2 mm. Preferably, atleast one or more cannulas are arranged, particularly preferably two,wherein one can be used for filling with a reactive liquid and anotherfor ventilation of the reaction space and for taking up surplus fluids.Such cannulas can be connected with the filling unit in a fixed or aninterchangeable manner, wherein preferably a connection, which cannot bedetached by the operator, for implementing disposable filling items isimplemented.

The filling unit can furthermore comprise a unit for covering thecannulas, so that any possible injury of the operator or contaminationof the environment can be avoided after separation of the systems.

Preferably, the filling unit furthermore comprises a suitable mechanicalinterface for snug-fit contacting of the reaction cartridge. Saidinterface can be developed, for example, in the form of specific snaps.In this manner, penetration of the seal of the chamber body at preferredsites can be ensured.

When processing the reaction cartridge in corresponding processingautomats, suitable mechanical measures are to be taken, which allowadjustment and accurate positioning in the devices. This particularlyapplies to the positioning for the replacement and/or the feeding ofliquids and the positioning of the reaction cartridge for detection ofthe signals after conduction of the reactions in the reaction chamber.

The device or the filling unit can furthermore comprise an integratedwaste container, which serves for taking up surplus or dislocatedgaseous or liquid media, like for example protective gas fillings orbuffers. The waste container can, for example, be filled with a furthergaseous, liquid, or solid medium, which binds the liquid or gaseoussubstances reversibly or irreversibly, like for example cellulose,filter materials, silica gels. In addition, the waste container can havea ventilation opening or can exhibit a negative pressure for improvingthe filling behavior of the entire unit.

Alternatively, the waste container can also be developed as separatemodule. In this case, the filling unit is equipped with correspondingfluid interfaces which can correspond to commercial standards, like forexample LuerLock, and which lead to the outside. Such interfaces canhave a form or force connection with continuing systems.

In a first special embodiment, filling is conducted by means of adetachable filling unit having an integrated waste container. Inparticular, the filling unit serves for non-recurrent filling of thereaction chamber. The filling unit is, for example, developed in such away that it is plugged or temporarily attached to the cartridge, thesamples are fed into the reaction space, and, after filling iscompleted, the filling unit is again separated from the cartridge and isdiscarded. In this special first embodiment, the filling unit furthercomprises an integrated waste container, which can be developed asdescribed above. An example for this embodiment is shown in FIG. 22. Theprocedure for filling a reaction cartridge by means of a modular fillingunit is shown in FIG. 23.

In a second special embodiment, filling is conducted by means of anintegrated filling unit. Herein, the filling unit is an integratedcomponent of the reaction cartridge and is therefore not separated fromthe latter; discarding the filling unit and the cartridge is conductedsimultaneously. Herein, the filling unit is preferably used fornon-recurrent filling of the reaction chamber and possibly for furtherprocess-internal fluid steps. In this embodiment, the filling unitfurthermore preferably comprises a technical device, which implements apreferred position of the cannulas in the system, in particular forpreventing inadvertent piercing of the cannulas into the seal of thechamber body. It is, however, also conceivable that the cannulas piercethe seal of the chamber body in said preferred position. Said technicaldevice can, for example, be implemented by means of establishingsprings, elastic elements, or specific recesses and bumps forimplementing a catch. In this embodiment, the filling unit furthercomprises a filling and waste channel, which comprises correspondingfluid interfaces, which can also correspond to commercial standards,like for example LuerLock, and which lead to the outside. Suchinterfaces can have a positive or non-positive interlocking withcontinuing systems and serve for feeding and/or removing gaseous and/orliquid media. An example for this embodiment is shown in FIG. 24. Theprocedure for filling a reaction cartridge having an integrated fillingunit is shown in FIG. 25.

In a third special embodiment, filling is conducted via an integratedfilling unit having an integrated waste container. In said embodiment,the filling unit is an integrated component of the reaction cartridgeand is therefore not separated from the latter; filling unit andcartridge are discarded simultaneously. Herein, the filling unit ispreferably used for non-recurrent filling of the reaction chamber andpossibly for further process-internal fluid steps.

In this embodiment, the filling unit furthermore preferably alsocomprises a technical device, which implements a preferred position ofthe cannulas in the system, preferably for preventing inadvertentpiercing of the cannulas into the seal of the chamber body. It is,however, also conceivable that the cannulas pierce the seal of thechamber body in said preferred position. Said technical device can, forexample, be implemented by means of establishing springs, elasticelements, or specific recesses and bumps for implementing a catch. Inthis embodiment, the filling unit furthermore comprises an integratedwaste container, which can be developed as described above. An examplefor this embodiment is shown in FIG. 26. The procedure for filling areaction cartridge with an integrated filling unit and integrated wastecontainer can, for example, be conducted by means of combining theprocedures described in FIGS. 23 and 25.

In the following, a special embodiment for arranging cannulas forpressure balance during the compression procedure will be described. Thecannulas of a filling tool for the cartridge can, for example, bearranged in such a way that both filling in a non-compressed state andtransfer of surplus reaction solutions during a compression of thereaction space is possible. This can preferably be achieved by means ofadapted construction of the seal and a cannula arrangement, wherein thecannulas preferably pierce the compensation regions within the reactionchamber. Such an arrangement is particularly suitable, if the surplusvolume cannot be taken up by means of a special seal design. An examplefor a possible vertical cannula arrangement with unaltered form of theseal is shown in FIG. 27.

The device according to the present invention can further comprise aunit, which is connected to the detection system, for controlling thetest procedure and/or for processing the signals recorded by means ofthe detection system. The controlling and/or processing unit can be amicro-controller or an industrial computer. This coupling of detectionunit and processing unit, which ensures the conversion of the reactionresults to the analysis result, allows, inter alia, the use of thedevice according to the present invention as hand-held device, forexample, in medical diagnostics.

In addition, the device according to the present invention furthermorepreferably has an interface for external computers. Inter alia, thisallows the transfer of data for external storage.

In a further preferred embodiment, the device is equipped with a coding,preferably a data matrix and/or a bar code, containing information onthe substance library and/or the conduction of the amplification and/ordetection reaction. By means of such an individual identificationnumber, the reading or detection device can automatically recognize,which test has been conducted. To this end, a data record containinginformation on the substance library, the conduction of the detectionreaction, and the like is stored in a database when manufacturing thedevice according to the present invention. Thus, the data record can, inparticular, contain information on the layout of the probes on the arrayand information as to how evaluation is to be conducted in the mostadvantageous manner. The data record or the data matrix can furthercontain information on the temperature-time regime of a PCR to beoptionally conducted for amplifying the target molecules. The datarecord thus obtained is preferably given a number, which is attached tothe holder in the form of the data matrix. Via the number recorded inthe data matrix, the set data record can then optionally be called whenreading out the substance library. Finally, the data matrix can be readout by the temperature controlling or regulating unit and othercontrollers, like for example a control for filling and unloading of thereaction chamber via the fluid containers, and an automatic conductionof amplification and detection reaction can thus be ensured.

The coding, like a data matrix, does not compellingly have to containthe entire information. It can also simply contain an identification oraccess number, by means of which the necessary data are then downloadedfrom a computer or a data carrier.

All preceding embodiments optionally have a displacement structurelocated on the second surface, as described above, namely on the side ofthe second surface that is facing the microarray. Thereby, thedisplacement structure is positioned in such a way that it is locatedopposite to the microarray and evenly rests on it in the compressedstate, wherein during compression the analyte solution being issubstantially displaced from the reaction chamber and the surface of themicroarray, respectively.

The device according to the present invention can be very easilymanufactured. In FIG. 3 it is shown that the process unit can consist ofonly four individual components, which are simply fit into one another.FIGS. 10 and 11 show embodiments, which can also be easily manufactureddue to the construction according to the present invention, althoughthey consist of several components. The geometric tolerances of thedimensions of the individual components can be very large with, forexample, 1/10 to 2/10 mm, so that, for example, the large-scaleinjection molding of seal and chamber body can be conducted in a verycost-efficient manner. The low tolerances are facilitated by means ofpressing the chip against the detection plane, as thereby the opticalpath to the detection microscope is hardly influenced by the componentsof the process unit. The only geometric quantities having a lowtolerance are the x,y-position of the chip and the thickness of thedetection plane. The variance of the z-position of the chip, however,only plays a subordinate part. Despite these low technical requirements,a focusing device at the optical system, for example a fluorescencedetection microscope, is not required. These properties clearly show thesuitability of the device according to the present invention for mobileon-site use. The preceding advantages also apply, if the devices havethe above-mentioned displacement structure.

In a further aspect of the present invention, a method for qualitativelyand/or quantitatively detecting molecular interactions between probe andtarget molecules is provided, which comprises the following steps:

-   -   a) introducing a sample, preferably a sample solution comprising        target molecules, into a reaction chamber of a device according        to the present invention as described above; and    -   b) detecting an interaction between the target molecules and the        probe molecules immobilized on the substrate.

The method according to the present invention allows the qualitativeand/or quantitative detection of molecular interactions between probeand target molecules in a reaction chamber, without necessitating areplacement of the sample or reaction liquids in order to remove adisturbing background after the interaction is completed and before thedetection.

Within the scope of the present invention, the detection of aninteraction between the probe and the target molecule is usuallyconducted as follows: Subsequently to fixing the probe or the probes toa specific matrix in the form of a microarray in a predetermined manneror subsequently to providing a microarray, the targets are contactedwith the probes in a solution and are incubated under definedconditions. As a result of the incubation, a specific interaction orhybridization occurs between probe and target. The bond occurring hereinis significantly more stable than the bond of target molecules toprobes, which are not specific for the target molecule.

The detection of the specific interaction between a target and its probecan be performed by means of a variety of methods, which normally dependon the type of the marker, which has been inserted into target moleculesbefore, during or after the interaction of the target molecule with themicroarray. Typically, such markers are fluorescent groups, so thatspecific target/probe interactions can be read outfluorescence-optically with high local resolution and, compared to otherconventional detection methods, in particular mass-sensitive methods,with little effort (see, for example, A. Marshall, J. Hodgson, DNAchips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31;G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16,40-44).

Depending on the substance library immobilized on the microarray and thechemical nature of the target molecules, interactions between nucleicacids and nucleic acids, between proteins and proteins, and betweennucleic acids and proteins can be examined by means of this testprinciple (for survey see F. Lottspeich, H. Zorbas, 1998, Bioanalytik,Spektrum Akademischer Verlag, Heidelberg/Berlin, Germany).

Herein, substance libraries, receptor libraries, peptide libraries, andnucleic acid libraries are considered as substance libraries, which canbe immobilized on microarrays or chips. The nucleic acid libraries takeby far the most important role, and include microarrays, on whichdeoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA)molecules are immobilized.

In a preferred embodiment of the method according to the presentinvention, before detection the distance between first and secondsurface is kept in a position, which allows processing of the samplesolution and/or the interaction between the target molecules and theprobe molecules immobilized on the substrate, for example amplificationof nucleic acids to be detected and/or hybridization between nucleicacids to be detected and the nucleic acid probes immobilized on thesubstrate, before detection in step b).

It is further preferred that in step b) the distance between the firstand the second surface is altered, preferably reduced. I.e. thedetection is preferably conducted with a reduced distance between atleast one area of the first surface, on which the detection has to takeplace and the probes can be immobilized, respectively, and detectionplane. Particularly preferably, the distance between first surface anddetection plane is about zero during detection.

In one embodiment, the microarray is guided towards the second surfacein order to reduce the distance between first and second surface.Preferably, this is ensured by pressing the first surface by applyingpressure exerted via at least one means for guiding the first surface,for example a tappet, a rod, a pin and/or a screw, wherein the pressurepoint of the means is located particularly below the microarray.

Pressing the first surface against the second surface or the detectionplane can be facilitated in that the first surface is elasticallydeformable at least in the region below the region onto which probes canbe immobilized or the detection of the targets can take place.Alternatively, the first surface can be developed by means of twosuperimposed layers, wherein one outer layer of the two superimposedlayers has a recess at least in the region below the region, onto whichthe probes can be immobilized or the detection of the targets can takeplace, and an inner layer of the two superimposed layers is formed by anelastic seal. Pressure is then exerted on the inner layer in thevicinity of the recess by the means for guiding the first surface.

The means for guiding the first surface, for example a pin, a rod, atappet and/or a screw, cannot only serve for exerting a pressure on thefirst surface, however. In the event that bubbles should form on the DNAchip, which would impede the detection, these bubbles can be removed bymeans of agitation by the means for guiding the first surface, forexample by means of a vibration frequency of about 20 Hz applied to thefirst surface, in particular in the form of an elastic membrane.

All preceding embodiments optionally have a displacement structurelocated on the second surface, as described above, namely on the side ofthe second surface that is facing the microarray. Thereby, thedisplacement structure is positioned in such a way that it is locatedopposite to the microarray and evenly rests on it in the compressedstate, wherein during compression the analyte solution being issubstantially displaced from the reaction chamber and the surface of themicroarray, respectively.

Furthermore, there is often the problem that the interaction, forexample the hybridization, at the chip surface takes a very long time.Among other reasons, this is due to the fact that the speed ofinteraction or hybridization is determined by diffusion. Preferably, theinteraction or hybridization speed can be increased by means ofagitation via the means for guiding the first surface, for example bymeans of a vibration frequency of about 20 Hz applied to the firstsurface, in particular in the form of an elastic membrane, as theagitation or vibration leads to mixing in the reaction chamber.

In a further embodiment, the second surface is guided towards the firstsurface in order to reduce the distance between the first and the secondsurface. In particular, this can be ensured in that the second surfaceis guided toward the first surface by means of pressure exerted on thesecond surface by the spacer.

In a further embodiment, the first surface is guided towards the secondsurface and the second surface is guided towards the first surface inorder to reduce the distance between the first and the second surface.In all preceding embodiments, the above-described displacementstructures may be present.

In the following, further embodiments for guiding the first surfacerelatively to the second surface or the second surface relatively to thefirst surface will be described. Said embodiments are not only suitablefor positioning the first surface or the region, onto which probes canbe immobilized or the detection of the targets can take place,relatively to the second surface or the detection surface, but can, inparticular, also be used for moving the probe array relatively to thedetection surface. By means of such a motion, for example an agitationof the solution in the reaction chamber can be achieved.

In one embodiment, the substrate, whereon probes can be immobilized orwhich represents the region where the detection of the targets issupposed to take place, and which, in this case, is not integrated inthe first surface, is moved relative to the detection surface or movedwithin the chamber by means of a magnetic field. The substrate and/orthe second surface, for example, contain a magnetic material or acomponent, whereto a magnetic material has been added, and/or is mountedin a holder consisting of an entirely or partially magnetic material. Itcan further be preferred that the substrate and/or the second surfaceare moved passively by moving a magnetic body, which is arranged belowthe respective surface and is, for example, connected with said surface,by means of a magnetic field.

In a further embodiment, the substrate is moved and/or positionedrelatively to the detection surface by means of gravitational impact.

In a further embodiment, the substrate is moved and/or positionedrelatively to the detection surface by means of a stream generated inthe reaction chamber. To this end, the device can, for example, bedeveloped in such a way that, in case the probe array is surrounded by aliquid stream, a negative pressure is generated at one side of thereaction chamber and a positive pressure is generated at the oppositeside, which leads to movement of the substrate in the reaction chamber.Such a stream can, for example, be implemented by means of thermalconvection, which is caused by local temperature differences in thechamber.

In a further embodiment, the substrate is moved and/or positionedrelatively to the detection surface by means of impact of an electricfield.

In a further embodiment, a gas bubble is generated below the probe arrayby means of local overheating, due to which the substrate is moved inthe chamber or is guided toward the detection surface. In the precedingembodiments, the above-mentioned displacement structures may be presentas well.

By means of reducing the distance between first and second surfacebefore the detection, the sample solution preferably is substantiallyentirely removed from the region between first surface and at least theregion of the first surface, onto which, for example, probes can beimmobilized either on the first surface or a substrate, and on whichdetection has to take place, and detection plane. Hereby, backgroundsignals, which are caused by labeled molecules, which are not bound tothe array surface, for example by labeled primers and/or labeled targetnucleic acids, which are not bound to the array surface, are reduced.

Thus, in the detection of step b), the distance between the first andthe second surface is preferably altered in such a way that the samplesolution between the first and the second surface is essentiallyremoved. The target-probe-complexes to be detected are then essentiallyeither located in increased concentration in the detection plane due toimmobilized probes or the size of the target-probe-complexes and adisturbing background is virtually avoided. This also applies in thepresence of a displacement structure.

In a further alternative embodiment, the first surface rests evenly onthe second surface forming the detection plane already in the originalstate of the device and is not only brought into the detection plane bymeans of guiding the first surface toward the second surface and/orguiding the second surface toward the first surface. In this embodiment,the first surface is not moistened by the sample solution during theprocessing steps. For conducting the interaction reaction, for example ahybridization, the first surface, which is preferably made of an elasticmaterial, for example an elastic membrane, is guided away from thedetection surface. Thereby, the chip surface is moved away from thedetection surface and is moistened by the sample solution. Theinteraction, for example a hybridization, can take place. For conductingthe detection and further processing, the first surface, for example inthe form of an elastic membrane, is released again, due to which itleaps back to its originally adjusted position, which can be acceleratedby means of pressure exerted by a means for guiding the first surface,for example a pin, a rod, a screw and/or a tappet. Thereby, the firstsurface is pressed towards the detection plane again and the detectioncan be conducted without having background. This also applies in thepresence of a displacement structure.

In a further embodiment of the method according to the presentinvention, a device according to the present invention, as describedabove, is used, the first surface of which is developed in a pivotablemanner around a rotation axis.

In a first position, which is also referred to as initial position, thesurface of the region, onto which for example probes can directly beimmobilized on a first surface or on a substrate and on which thetargets have to be detected (e.g., a microarray), rests essentiallyevenly on the second surface, i.e. the substrate surface with the probemolecules immobilized thereon is essentially not moistened by the samplesolution. In the space formed in the first position between the secondflanking portion of the first surface and the second surface, theprocessing chamber, the processing of the reaction solution ispreferably conducted, i.e. in particular purification, re-concentration,washing and rinsing and/or amplification steps.

Subsequently, the pivotable first surface is brought to a secondposition, wherein the first surface is arranged relatively to the secondsurface at an angle other than 180°, preferably at an angle of 45°.Preferably, this is conducted by means of traction exerted on the firstflanking portion of the first surface and/or pressure exerted on thesecond flanking portion of the first surface by means of a means forguiding the first surface, as described above. By means of guiding thefirst surface to the second position, the microarray is guided away fromthe second surface and the sample solution penetrates the cavity formingbetween microarray and second surface. The probe molecules immobilizedon the substrate of the microarray are freely accessible for the targetmolecules present in the sample solution, so that an interactionreaction between probe and target molecules can occur. In thisembodiment of the method according to the present invention, pressureand/or traction exerted on the first surface has the advantage that, inthis manner, the sample solution is moved and thus the interactionreaction can be accelerated.

For conducting the detection and, optionally, further processing, thepivotable first surface is guided back to the first position, forexample by means of pressure exerted on the first flanking portion ofthe first surface and/or traction exerted on the second flanking portionof the first surface or, in the case of elastic development of the firstsurface, by means of releasing the first flanking portion. Now, thementioned region of the first surface again rests essentially evenly onthe second surface, so that the sample solution between the secondsurface and the microarray is essentially displaced in this position andan essentially background-free detection can take place. The precedingembodiment may also comprise a displacement structure.

The targets to be examined can be present in any kind of sample,preferably in a biological sample.

Preferably, the targets are isolated, purified, copied, and/or amplifiedbefore their detection and quantification by means of the methodaccording to the present invention.

The method according to the present invention further allows theamplification and the qualitative and/or quantitative detection ofnucleic acids in a reaction chamber, wherein the detection of molecularinteractions or hybridizations can be conducted after completion of acyclic amplification reaction without necessitating replacement of thesample or reaction liquids. The method according to the presentinvention further also ensures a cyclic detection of hybridizationevents in an amplification, i.e. a detection of the hybridization evenduring the cyclic amplification reaction. Finally, with the aid of themethod according to the present invention, the amplification productscan be quantified during the amplification reaction and after completionof the amplification reaction.

Usually, the amplification is performed by means of conventional PCRmethods or by means of a method for the parallel performance ofamplification of the target molecules to be analyzed by means of PCR anddetection by means of hybridization of the target molecules with thesubstance library support, as is described above.

In a further embodiment, the amplification is performed as a multiplexPCR in a two-step process (see also WO 97/45559). In a first step, amultiplex PCR is performed by means of using fusion primers, whose3′-ends are gene specific and whose 5′-ends represent a universalregion. The latter is the same in all forward and reverse primers usedin the multiplex reaction. In this first stage, the amount of primer islimiting. Hereby, all multiplex products can be amplified until auniform molar level is achieved, given that the number of cycles isadequate for reaching primer limitation for all products. In a secondstage, universal primers identical to the 5′-regions of the fusionprimers are present. Amplification is performed until the desired amountof DNA is obtained.

In a further preferred embodiment of the method according to the presentinvention, detection is performed during the cyclic amplificationreaction and/or after completion of the cyclic amplification reaction.Preferably, detection is performed during the amplification reaction, inevery amplification cycle. Alternatively, detection can also bedetermined in every second cycle or every third cycle or in anyarbitrary intervals.

In the conduction of a linear amplification reaction, wherein the targetamount increases by a certain amount with each step, or an exponentialamplification reaction, for example a PCR, wherein the DNA target amountmultiplies with each step, in the process unit, the chip can thus bepressed towards the detection plane after every amplification step andtherefore the detection can be conducted. It is thus possible to performon-line surveillance of the amplification reaction. In particular in thecase of non-linear amplification reactions, it is thereby possible todetermine the initial concentration of the DNA target amount. In thecase of protein-targets and -probes, for example, binding kinetics canbe detected in an analogous manner as already mentioned above.

In this manner, the number of amplification steps can furthermore beoptimized on-line. As soon as the DNA target amount has reached aspecific concentration, the amplification is discontinued. If theinitial target concentration is low, the number of amplification stepsis increased in order to be able to conduct an assured analysis of theproducts. In the case of reduced reaction time of positive controls, theanalysis process can be discontinued very early.

The chemicals necessary for conducting an amplification reaction, likefor example polymerase, buffer, magnesium chloride, primers, labeled, inparticular fluorescence-labeled primers, dNTPs and the like, can beprovided in the reaction chamber, for example in freeze-dried form.

Preferably, the cyclic amplification reaction is a PCR. In a PCR, threetemperatures for each PCR cycle are usually passed through. Preferably,the hybridized nucleic acids detach from the microarray at the highesttemperature, i.e. the denaturation temperature. A preferred value forthe denaturation temperature is 95° C. Therefore, a hybridizationsignal, which serves as zero value or reference value for the nucleicacids detected in the respective PCR cycle, can be determined at thisdenaturation temperature.

At the temperature following in the PCR cycle, an annealing temperatureof, for example, about 60° C., a hybridization between the nucleic acidsto be detected and the nucleic acids immobilized on the substrate of themicroarray is facilitated. Therefore, in one embodiment of the methodaccording to the present invention, the detection of target nucleicacids present in a PCR cycle is performed at the annealing temperature.

In order to enhance the sensitivity of the method according to thepresent invention, it can further be advantageous to lower thetemperature below the annealing temperature, so that the detection ispreferably performed at a temperature below the annealing temperature ofan amplification cycle. For example, the detection can be performed at atemperature in a range of 25° C. to 50° C. and preferably in a range of30° C. to 45° C.

In a further alternative embodiment of the method according to thepresent invention, the hybridization between nucleic acids to bedetected and the nucleic acids immobilized on the substrate of themicroarray is at first performed at a low temperature, in order tosubsequently raise the hybridization temperature. Such an embodiment hasthe advantage that the hybridization time is reduced compared tohybridizations at temperatures of more than 50° C. without losingspecificity in the interactions.

If the zero value or reference value determined at denaturationtemperature is subtracted from the measured value determined at or belowthe annealing temperature, a measured result free of disturbances, inwhich fluctuation and drift are eliminated, can be obtained.

Usually, the target molecules to be detected are equipped with adetectable marker. In the method according to the present invention, thedetection is thus preferably conducted by means of equipping the boundtargets with at least one label, which is detected in step b).

As already mentioned above, the label coupled to the targets or probespreferably is a detectable unit or a detectable unit coupled to thetargets or probes via an anchor group. With respect to the possibilitiesfor detection or labeling, the method according to the present inventionis very flexible. Thus, the method according to the present invention iscompatible with a variety of physical, chemical, or biochemicaldetection methods. The only prerequisite is that the unit or structureto be detected can directly be coupled or can be linked via an anchorgroup, which can be coupled with the oligonucleotide, to a probe or atarget, for example an oligonucleotide.

The detection of the label can be based on fluorescence, magnetism,charge, mass, affinity, enzymatic activity, reactivity, a gold label,and the like. Thus, the label can, for example, be based on the use offluorophore-labeled structures or components. In connection withfluorescence detection, the label can be an arbitrary dye, which can becoupled to targets or probes during or after their synthesis. Examplesare Cy dyes (Amersham Pharmacia Biotech, Uppsala, Sweden), Alexa dyes,Texas Red, Fluorescein, Rhodamin (Molecular Probes, Eugene, Oreg., USA),lanthanides like samarium, ytterbium, and europium (EG&G, Wallac,Freiburg, Germany).

Particularly preferably, said detectable marker is a fluorescencemarker. As already mentioned above, the use of the device according tothe present invention in the method according to the present inventionensures the detection of the fluorescence markers by means of afluorescence microscope without autofocus, for example a fluorescencemicroscope with fixed focus.

Apart from fluorescence markers, luminescence markers, metal markers,enzyme markers, radioactive markers, and/or polymeric markers can alsobe used within the scope of the present invention as labeling and/ordetection unit, which is coupled to the targets or the probes.

Likewise, a nucleic acid, which can be detected by means ofhybridization with a labeled reporter (sandwich hybridization), can beused as label (tag). Diverse molecular biological detection reactionslike primer extension, ligation, and RCA are used for the detection ofthe tag.

In an alternative embodiment of the method according to the presentinvention, the detectable unit is coupled with the targets or probes viaan anchor group. Preferably used anchor groups are biotin, digoxigenin,and the like. In a subsequent reaction, the anchor group is converted bymeans of specifically binding components, for example streptavidinconjugates or antibody conjugates, which in turn are detectable ortrigger a detectable reaction. With the use of anchor groups, theconversion of the anchor groups to detectable units can be performedbefore, during, or after the addition of the sample comprising thetargets, or, optionally, before, during, or after the cleavage of aselectively cleavable bond in the probes. Such selectively cleavablebonds in the probes are, for example, described in the InternationalPatent Application WO 03/018838, the relevant contents of which arehereby explicitly referred to.

According to the present invention, labeling can also be performed bymeans of interaction of a labeled molecule with the probe molecules. Forexample, labeling can be performed by means of hybridization of anoligonucleotide labeled as described above with an oligonucleotide probeor an oligonucleotide target.

Further labeling methods and detection systems suitable within the scopeof the present invention are described, for example, in Lottspeich andZorbas, Bioanalytik, Spektrum Akademischer Verlag, Heidelberg, Berlin,Germany 1998, chapter 23.3 and 23.4.

In a preferred embodiment of the method according to the presentinvention, detection methods are used, which in result yield an adducthaving a particular solubility product, which leads to a precipitation.For labeling, in particular substrates or educts are used, which can beconverted to a hardly soluble, usually stained product. In this labelingreaction, for example, enzymes can be used, which catalyze theconversion of a substrate to a hardly soluble product. Reactionssuitable for leading to a precipitation at the array elements as well aspossibilities for the detection of the precipitation are, for example,described in the International Patent Application WO 00/72018 and in theInternational Patent Application WO 02/02810, whose relevant contentsare hereby explicitly referred to.

In a particularly preferred embodiment of the method according to thepresent invention, the bound targets are equipped with a labelcatalyzing the reaction of a soluble substrate or educt to form a hardlysoluble precipitation at the array element, where a probe/targetinteraction has occurred or acting as a crystal nucleus for theconversion of a soluble substrate or educt to a hardly solubleprecipitation at the array element, where a probe/target interaction hasoccurred.

In this manner, the use of the method according to the present inventionallows for the simultaneous qualitative and quantitative analysis of avariety of probe/target interactions, wherein several array elementshaving a size of ≤1000 μm, preferably of ≤100 μm, and particularlypreferably of ≤50 μm may be realized.

The use of enzymatic labels is known in immunocytochemistry and inimmunological tests based on microtiter plates (see E. Lidell and I.Weeks, Antibody Technology, BIOS Scientific Publishers Limited, 1995).Thus, for example, enzymes catalyze the conversion of a substrate to ahardly soluble, usually stained product.

Particularly preferably, the reaction leading to precipitation formationat the array elements is a conversion of a soluble substrate or educt toa hardly soluble product, catalyzed by an enzyme. In a specialembodiment, the reaction leading to precipitation formation at the arrayelements is an oxidation of 3,3′,5,5′-tetramethylbenzidine, catalyzed bya peroxidase.

Preferably, horseradish peroxidase is used for the oxidation of3,3′,5,5′-tetramethylbenzidine. However, additional peroxidases areknown by the skilled person, which can be used for the oxidation of3,3′,5,5′-tetramethylbenzidine.

3,3′,5,5′-tetramethylbenzidine, when exposed to a peroxidase, is assumedto be oxidized in a first step to form a blue-stained radical cation(see, for example, Gallati and Pracht, J. Clin. Chem. Clin. Biochem.1985, 23, 8, 454). This blue-stained radical cation is precipitated inform of a complex by using a polyanion, such as dextran sulfate. Theprecipitation reaction by means of peroxidase-catalyzed oxidation of3,3′,5,5′-tetramethylbenzidine is, for example, described in EP 0 456782.

Without any intention of being complete, the following Table 1summarizes several reactions that are suitable to cause a precipitationat array elements, where an interaction between target and probe hasoccurred:

TABLE 1 catalyst or substrate or educt crystal nucleus horseradishperoxidase DAB (3,3′-diaminobenzidine) 4-CN (4-chloro-1-naphthol) AEC(3-amino-9-ethylcarbazole) HYR (p-phenylenediamine-HCl and pyrocatechol)TMB (3,3′,5,5′-tetramethylbenzidine) naphthol/pyronin alkalinephosphatase brom-chlor-indolyl-phosphate (BCIP) and nitrobluetetrazolium (NBT) glucose oxidase t-NBT and m-PMS (nitroblue tetrazoliumchloride and phenazine methosulfate gold particles silver nitrate silvertartrate

In particular, the detection of probe/target interactions via insolubleprecipitates is described in WO02/02810.

In the following, embodiments of the present invention are described,which can serve to overcome problems likely to arise in the detection ofmolecular interactions on solid supports, such as for preventing thepossible formation of Newton's rings between detection surface and probearray.

The manifestation of Newton's rings is essentially determined by thetype of illumination, the wavelength of the light used for detection,the distance between detection plane and probe array, and the refractionindex of the solution located in the chamber. Such Newton's rings can,for example, be prevented by means of altering the wavelength of thelight used for detection, by using a solution having the same or asimilar refraction index as the detection surface and/or the probearray, and/or by using an immersion liquid between detection surface andprobe array.

Furthermore, Newton's rings can be prevented by means of applyingspacers on the chip or on the regions on the side of the detectionsurface facing the chip.

Furthermore, Newton's rings can be prevented by means of applying theprobe array onto a rough support surface.

Furthermore, Newton's rings can be prevented by means of applying theprobe array onto a light-absorbing surface.

As a further possibility the contact pressure by which the first surfaceis guided relatively to the detection surface may be permanently variedduring detection. Thus, the thickness of the gap between chip anddetection surface, and therefore also the position of Newton's rings, isaltered. By integrating the fluorescence signal to be detected overtime, a falsification of the measured values of the spots in relation toeach other is prevented.

It is a further particularly preferably possibility of preventingNewton's rings to use several light sources from different directionsfor illuminating and therefore agitating the fluorophores of the boundtargets.

Background fluorescence caused by fluorophores of unbound targets in thedisplaced liquid can lead to distortion of the signal detected. This canpreferably be prevented by means of using an aperture, which is, forexample, mounted on the detection surface or the chip and/or the regionsaround the chip or the imaging optics, and is configured in such a waythat only the surface of the probe array is illuminated or imaged.

By using appropriate light sources, such as lasers, illumination may beinhomogenous due to coherence of the light. Such inhomogeneities can bereduced or prevented by using waveguides and/or combining filters and/orlight of different wavelengths. Likewise, movement of the light sourcein order to eliminate such effects is also conceivable.

By using an organic or inorganic light-absorbing layer, which isnon-fluorescent in the selected wavelength range, on the substrate ofthe probe array, the fluorescence background signal caused by the arraysupport and/or elements located behind the same, can be reduced orprevented. Preferably, a black chromium layer is employed as protectivelayer.

In all above-described embodiments of the inventive method, apre-amplification of the material to be analyzed is not required. Fromthe sample material extracted from bacteria, blood, or other cells,specific partitions can be amplified using a PCR (polymerase chainreaction), in particular in the presence of the inventive device or thesubstance library support, as is described in DE 102 53 966, andhybridized to the support. This represents a substantial reduction oflabor effort.

Thus, the method according to the present invention is particularlysuitable for the parallel amplification of the target molecules to beanalyzed by PCR and the detection by hybridization of the targetmolecules with the substance library support. There, the nucleic acid tobe detected is first amplified by PCR, wherein preferably at least onecompetitor inhibiting the formation of one of the two template strandsamplified by PCR is initially added to the reaction. In particular, aDNA molecule, which competes with one of the primers used for the PCRamplification of the template for binding to the template and which cannot be extended enzymatically, is added to the PCR. The single-strandednucleic acid molecules amplified by PCR are then detected by means ofhybridization with a complementary probe. Alternatively, the nucleicacid to be detected is first amplified using a surplus of single strandby PCR and is detected by means of a subsequent hybridization with acomplementary probe, wherein a competitor, which is a DNA molecule or amolecule of a nucleic acid analog capable of hybridizing to one of thetwo strands of the template but not to the region detected byhybridization to a probe and which cannot be enzymatically extended, isinitially added to the PCR reaction.

Any molecule causing a preferred amplification of only one of the twotemplate strands present in the PCR reaction can be used as competitorin the PCR. Thus, according to the present invention, competitors can beproteins, peptides, DNA ligands, intercalators, nucleic acids or analogsthereof. Proteins or peptides, which are capable of bindingsingle-stranded nucleic acids with sequence specificity and which havethe above-defined properties, are preferably used as competitors.Particularly preferably, nucleic acid molecules and nucleic acid analogmolecules are used as to break open secondary structures.

The formation of one of the two template strands is substantiallyinhibited by the initial addition of the competitor to the PCR duringamplification. “Substantially inhibited” means that a surplus of singlestrand and a amount of the other template strand sufficient to allow anefficient detection of the amplified strand by means of hybridizationare produced in the PCR. Therefore, the amplification does not followexponential kinetics of the form 2^(n) (with n=number of cycles), butrather attenuated amplification kinetics of the form <2^(n).

The single strand surplus obtained by means of the PCR in relation tothe non-amplified strand has the factor 1.1 to 1,000, preferably thefactor 1.1 to 300, also preferably the factor 1.1 to 100, particularlypreferably the factor 1.5 to 100, also particularly preferably thefactor 1.5 to 50, in particular preferably the factor 1.5 to 20, andmost preferably the factor 1.5 to 10.

Typically, the function of a competitor will be to bind selectively toone of the two template strands and therefore to inhibit theamplification of the corresponding complementary strand. Therefore,competitors can be single-stranded DNA- or RNA-binding proteins havingspecificity for one of the two template strands to be amplified in aPCR. They can also be aptamers sequence-specifically binding only tospecific regions of one of the two template strands to be amplified.

Preferably, nucleic acids or nucleic acid analogs are used ascompetitors. Usually, the nucleic acids or nucleic acid analogs will actas competitors of the PCR either by competing with one of the primersused for the PCR for the primer binding site or by being capable ofhybridizing with a region of a template strand to be detected due to asequence complementarity. This region is not the sequence detected bythe probe. Such nucleic acid competitors are enzymatically notextendable.

The nucleic acid analogs can, for example, be so-called peptide nucleicacids (PNA). However, nucleic acid analogs can also be nucleic acidmolecules, in which the nucleotides are linked to one another via aphosphothioate bond instead of a phosphate bond. They can also benucleic acid analogs, wherein the naturally occurring sugar componentsribose or deoxyribose have been replaced with alternative sugars, likefor example arabinose or trehalose or the like. Furthermore, the nucleicacid derivative can be “locked nucleic acid” (LNA). Further nucleic acidanalogs are known to the person skilled in the art.

Preferably, DNA or RNA molecules, and particularly preferably DNA or RNAoligonucleotides or their analogs, are used as competitors.

Depending on the sequence of the nucleic acid molecules or nucleic acidanalogs used as competitors, the inhibition of the amplification of oneof the two template strands within the scope of the PCR reaction isbased on different mechanisms. In the following, this is exemplarilydiscussed for a DNA molecule.

If, for example, a DNA molecule is used as competitor, it may have asequence, which is at least partially identical to the sequence of oneof the primers used for the PCR such that a specific hybridization ofthe DNA competitor molecule with the corresponding template strand ispossible under stringent conditions. Since, within the presentinvention, the DNA molecule used for competition in this case is notextendable by means of a DNA polymerase, the DNA molecule competes withthe respective primer for binding to the template during the PCRreaction. Depending on the ratio of the DNA competitor molecule and theprimer, the amplification of the template strand defined by the primercan thus be inhibited in such a way that the production of this templatestrand is significantly reduced. Thereby, the PCR proceeds according toexponential kinetics higher than would be expected with the amounts ofcompetitors used. In this manner, a single strand surplus emerges in anamount, which is sufficient for the efficient detection of the amplifiedtarget molecules by means of hybridization.

In this embodiment, the nucleic acid molecules or nucleic acid analogsused for competition must not be enzymatically extendable.“Enzymatically not extendable” means that the DNA or RNA polymerase usedfor the amplification cannot use the nucleic acid competitor as primer,i.e. it is not capable of synthesizing the corresponding opposite strandof the template 3′ from the sequence defined by the competitor.

Alternatively to the above-depicted possibility, the DNA competitormolecule can also have a sequence complementary to a region of thetemplate strand to be detected, which is not addressed by one of theprimer sequences and which is enzymatically not extendable. In a PCR,the DNA competitor molecule will then hybridize to this template strandand correspondingly block the amplification of this strand.

The person skilled in the art knows that the sequences of DNA competitormolecules or, in general, nucleic acid competitor molecules can beselected appropriately. If the nucleic acid competitor molecules have asequence, which is not substantially identical to the sequence of one ofthe primers used for the PCR, but is complementary to another region ofthe template strand to be detected, this sequence is to be selected insuch a way that it does not fall within the region of the templatesequence, which is detected with a probe within the scope of thehybridization. This is necessary because there does not have to occur aprocessing reaction between the PCR and the hybridization reaction. If anucleic acid molecule, which falls within the region to be detected,were used as competitor, it would compete for binding to the probeagainst the single-stranded target molecule.

Such competitors preferably hybridize close to the template sequencedetected by the probe. According to the present invention, the positionspecification “close to” is to be understood in the same way as givenfor agents breaking open secondary structures. However, the competitorsaccording to the present invention can also hybridize in the immediateproximity of the sequence to be detected, i.e. at exactly onenucleotide's distance from the target sequence to be detected.

If nucleic acids or nucleic acid analogs that are not enzymaticallyextendable are used as competing molecules, they are to be selected withrespect to their sequence and structure in such a way that they cannotbe enzymatically extended by DNA or RNA polymerases. Preferably, the3′-end of a nucleic acid competitor is designed in such a way that ithas no complementarity to the template and/or has at its 3′-end asubstituent other than the 3′—OH group.

If the 3′ end of the nucleic acid competitor has no complementarity tothe template, regardless of whether the nucleic acid competitor binds toone of the primer binding sites of the template or to one of thesequences of the template to be amplified by means of the PCR, thenucleic acid competitor cannot be extended by the conventional DNApolymerases due to the lack of base complementarity at its 3′-end. Thistype of non-extensibility of nucleic acid competitors by DNA polymerasesis known to the person skilled in the art. Preferably, the nucleic acidcompetitor has no complementarity to its target sequence at its 3′-endwith respect to the last 4 bases, particularly preferably to the last 3bases, in particular preferably to the last 2 bases, and most preferablyto the last base. In the mentioned positions, such competitors can alsohave non-natural bases, which do not allow hybridization.

Nucleic acid competitors, which are enzymatically not extendable, canalso have a 100% complementarity to their target sequence, if they aremodified in their backbone or at their 3′-end in such a way that theyare enzymatically not extendable.

If the nucleic acid competitor has at its 3′-end a group other than theOH group, these substituents are preferably a phosphate group, ahydrogen atom (dideoxynucleotide), a biotin group, or an amino group.These groups cannot be extended by conventional polymerases.

The use of a DNA molecule, which competes with one of the two primersused for the PCR for binding to the template, and which was providedwith an amino linkage at its 3′-end during chemical synthesis, as acompetitor in such a method is particularly preferred. Such competitorscan have 100% complementary to their target sequence.

However, nucleic acid analog competitors, like for example PNAs do nothave to have a blocked 3′—OH group or a non-complementary base at their3′-end as they are not recognized by the DNA polymerases because of thebackbone modified by the peptide bond and thus are not extended. Othercorresponding modifications of the phosphate group, which are notrecognized by the DNA polymerases, are known to the person skilled inthe art. Among those are inter alia nucleic acids having backbonemodifications, like for example 2′-5′ amide bonds (Chan et al. (1999) J.Chem. Soc., Perkin Trans. 1, 315-320), sulfide bonds (Kawai et al.(1993) Nucleic Acids Res., 1 (6), 1473-1479), LNA (Sorensen et al.(2002) J. Am. Chem. Soc., 124 (10), 2164-2176) and TNA (Schoning et al.(2000) Science, 290 (5495), 1347-1351).

Several competitors hybridizing to different regions of the template(for example, inter alia, the primer binding site) can alsosimultaneously be used in a PCR. The efficiency of the hybridization canadditionally be increased, if the competitors have properties ofsecondary structure breakers.

In an alternative embodiment, the DNA competitor molecule can also havea sequence complementary to one of the primers. Depending on the ratioof antisense DNA competitor molecule and primer, such, for example,antisense DNA competitor molecules can then be used to titrate theprimer in the PCR reaction, so that it will no longer hybridize with thecorresponding template strand and, correspondingly, only the templatestrand defined by the other primer is amplified. The person skilled inthe art is aware of the fact that, in this embodiment of the invention,the nucleic acid competitor can, but does not have to, be enzymaticallyextendable.

If, within the present invention, it is referred to nucleic acidcompetitors, this includes nucleic acid analog competitors, unless adifferent meaning arises from the respective context. The nucleic acidcompetitor can bind to the corresponding strand of the templatereversibly or irreversibly. The bond can take place via covalent ornon-covalent interactions.

Preferably, binding of the nucleic acid competitor takes place vianon-covalent interactions and is reversible. In particular preferably,binding to the template takes place via formation of Watson-Crick basepairings.

The sequences of the nucleic acid competitors normally adapt to thesequence of the template strand to be detected. In the case of antisenseprimers, though, they adapt to the primer sequences to be titrated,which are in turn defined by the template sequences, however.

PCR amplification of nucleic acids is a standard laboratory method, thevarious possibilities of variation and development of which are familiarto the person skilled in the art. In principle, a PCR is characterizedin that the double-stranded nucleic acid template, usually adouble-stranded DNA molecule, is first subjected to heat denaturationfor 5 minutes at 95° C., whereby the two strands are separated from eachother. After cooling down to the so-called “annealing” temperature(defined by the primer with the lower melting temperature), the forwardand reverse primers present in the reaction solution accumulate at thosesites in the respective template strands, which are complementary totheir own sequence. Herein, the “annealing” temperature of the primersadapts to the length and base composition of the primers. It can becalculated on the basis of theoretical considerations. Information onthe calculation of “annealing” temperatures can be found, for example,in Sambrook et al. (vide supra).

Annealing of the primers, which is typically performed in a range oftemperatures between 40 to 75° C., preferably between 45 to 72° C. andin particular preferably between 50 to 72° C., is followed by anelongation step, wherein deoxyribonucleotides are linked with the 3′-endof the primers by the activity of the DNA polymerase present in thereaction solution. Herein, the identity of the inserted dNTPs depends onthe sequence of the template strand hybridized with the primer. Asnormally thermostable DNA polymerases are used, the elongation stepusually runs at between 68 to 72° C.

In a symmetrical PCR, an exponential amplification of the nucleic acidregion of the target defined by the primer sequences is achieved bymeans of repeating the described cycle of denaturation, annealing andelongation of the primers. With respect to the buffer conditions of thePCR, the usable DNA polymerases, the production of double-stranded DNAtemplates, the design of primers, the selection of the annealingtemperature, and variations of the classic PCR, the person skilled inthe art has numerous references at his disposal.

The person skilled in the art is familiar with the fact that, forexample, single-stranded RNA, such as mRNA, can be used as template aswell. Usually, this mRNA is previously transcribed into adouble-stranded cDNA via a reverse transcription.

In a preferred embodiment, a thermostable DNA-dependent polymerase isused as polymerase. In a particularly preferred embodiment, athermostable DNA-dependent DNA polymerase is used, which is selectedfrom the group consisting of Taq-DNA polymerase (Eppendorf, Hamburg,Germany and Qiagen, Hilden, Germany), Pfu-DNA polymerase (Stratagene, LaJolla, USA), Tth-DNA polymerase (Biozym Epicenter Technol., Madison,USA), Vent-DNA polymerase, DeepVent-DNA polymerase (New England Biolabs,Beverly, USA), Expand-DNA polymerase (Roche, Mannheim, Germany).

The use of polymerases, which have been optimized from naturallyoccurring polymerases by means of specific or evolutive alteration, isalso preferred. When performing the PCR in the presence of the substancelibrary support, the use of the Taq-polymerase by Eppendorf (Germany)and of the Advantage cDNA Polymerase Mix by Clontech (Palo Alto, Calif.,USA) is particularly preferred.

All devices according to the present invention, which can be employedfor the methods according to the present invention, can, in a preferredembodiment, have a so-called displacement structure, irrespective ofwhether they have or have not immobilized probe molecules in a region ofthe first surface, and irrespective of whether they are used, forexample, for nucleic acid target/probe interactions or proteintarget/probe interactions or for concentration measurements of targetswithout probes.

Any preceding methods may also be performed using an inventive devicehaving a displacement structure, as described above.

Another aspect of the present invention relates to the use of aninventive device for performing microarray-based tests.

In the following, special embodiments of the inventive devices and theinventive method are described.

In FIG. 5, it is shown that the first surface, here an elastic membrane,in which preferably a heating device is integrated, is deformed by meansof a pin or a tappet and that the chip is thus pressed towards thedetection surface. Furthermore, the detection surface is pressed intothe reaction chamber via a spacer on the second surface and thusapproaches the DNA chip from above until the liquid between DNA chip anddetection plane is almost entirely displaced. The elastic seals sealingthe reaction chamber are compressed by guiding the detection surfacetowards the chip. The displaced fluid deforms the seal in such a waythat the air is compressed in air compensation chambers. This occurs ina more efficient manner, if the second surface has the displacementstructure described above.

However, the process unit can also be configured such that either onlythe first surface, for example in form of an elastic membrane, isdeformed or only the detection plane is pressed into the chamber,potentially by using a displacement structure.

In FIG. 6, a further technical embodiment for compressing the processunit is depicted. The reaction chamber is sealed laterally and at theside opposite the detection surfacee by a sealing membrane, on which aDNA chip is attached. At the level of the DNA chip, the sealing membraneseals a hole in the lower side of the chamber body. The hole is slightlysmaller than the DNA chip. When conducting a PCR in the reactionchamber, the hole is tightly sealed by the internal pressure forming dueto the raised temperatures connected with the PCR. Therefore, despitethe labile sealing membrane, the chamber is pressure-proof (principle ofthe self-closing valve). For detection, a pin or a tappet is pushedthrough the lower side hole. The sealing membrane is lifted and the DNAchip is pressed against the detection plane. In order to ensure therequired elasticity of the sealing membrane, the membrane can beprovided with a compensation fold. In this embodiment, the pressurecompensation chambers are also compressed by the displaced liquid. Thisembodiment may also have a displacement structure.

The following examples are for the purpose of illustrating theinvention, and are not to be construed as limiting the scope of theinvention.

EXAMPLES Example 1: Setup of a Reaction Cartridge without an IntegratedHeating

In FIGS. 8 and 9, an embodiment of a processing unit without integratedheating and a device for guiding a substrate which overlies the firstsurface and onto which the probes can be immobilized toward thedetection plane are depicted. The substrate in the device shown can beread out by means of a conventional fluorescence microscope (for exampleAxioskop, Zeiss, Jena, Germany).

Example 2: Setup of a Reaction Cartridge Having a Silicon HeatingSubstrate

The variant of the processing unit of the device according to thepresent invention, which is shown in FIGS. 10 and 11, is a miniaturizedreaction cartridge having a substrate overlying the first substratewhich is represented by a heating/sensor substrate and onto which probescan be immobilized wherein it is not about a microarray, a siliconheating substrate having an integrated temperature sensor (“heatingsubstrate”) for adjusting distinctive temperatures in the reactionchamber as well as a circuit board optionally having an EPROM forelectrically contacting the heating substrate. The individual componentsare embedded in two shells made of synthetic material. The entire unitis a spatially closed system, in which all required reactions (forexample PCR) can be conducted in a temperature-controlled manner.

First, the circuit board is inserted into the provided shaft in thelower shell (with the EPROM facing downward). On the upper side of thecircuit board, three electric contact pads are arranged, which ensurethe electric connection with the subsequently inserted heatingsubstrate, which in turn bears the contact pads. Said heating substratehas a size of 8 mm×6 mm and a thickness of about 0.6 mm. The heatingsubstrate ensures exact adjustment of different temperatures (forexample of 40° C. to 95° C.) within the scope of the examinationconducted. Herein, measuring the temperature in the reaction chamber canbe conducted either via the sensor integrated in the heating substrateor via an external measuring unit, which measures the temperaturedirectly on the surface of the heating substrate. In the latter case,the integrated sensor in the heating substrate can be omitted. Theintegrated components used for heating and/or temperature measurementcan, for example, be diodes or also transistors. The surface of thesilicon heating substrate, which is facing toward the reaction space,contains no electric systems whatsoever and is coated with an SiO₂passivating layer.

The next component is an elastic seal, which laterally limits thereaction space.

In the center of the reaction space, the substrate is attached in such away that it is facing toward the detection plane. After inserting thedetection plane in the form of a glass surface, said surface stillprotrudes from the lower shell by 0.2 mm. By subsequently joining theupper shell, which is guided by means of locating pins, the glasssurface is pressed against the seal and thus ensures optimal sealing ofthe reaction chamber.

Subsequently, the reaction chamber can be filled with reaction solution.Herein, it is to be noted that only the inner space containing the chipis filled, but not the outer chambers. The liquids required are injectedinto the reaction space with cannulas via the provided cannula guide.

Subsequently, biochemical reactions controlled via the silicon heatingsubstrate, like for example PCR and/or hybridization, can be conductedin the reaction chamber.

For detecting the intermediate results or the final result, thedetection plane is pressed against the substrate from above by means ofthe spacers of the detection unit, until the distance between detectionplane and substrate is about zero. Herein, the surrounding liquid isdislocated into the outer chambers, where it compresses the local air.This process is reversible and can, for example, be conducted after eachPCR cycle. The same is true for binding assays between protein targetsand protein probes.

Due to its compact design and the internal circuit board having an EPROMand the integrated heating substrate, this variant of the deviceaccording to the present invention is particularly suitable for mobileuse.

Example 3: Detection of the Decrease of Background Signal by Displacingthe Analyte

All fluorescence measurements described in this example were performedusing a fluorescence microscope (Zeiss, Jena, Germany). Excitationoccurred in incident light using a white light source and a filter setsuitable for Cyanine 3. The signals were recorded by means of a CCDcamera (PCO-Sensicam, Kehlheim, Germany). In the following, thethickness of the gap denotes the distance between microarray anddetection plane.

a) Measuring the Fluorescence Signal of the Analyte Depending on theThickness of the Gap

Channel shells having defined channel depth (5 μm, 10 μm, 28 μm) made ofSylgard were cast. The channels had a width of 125 μm. A glass chip wasplaced across the unequally deep channels. The channels were then filledwith a 200 nM solution of a Cy3-labeled oligonucleotide in 2×SSC+0.2%SDS and the signal was measured with an exposure time of 1.5 s.

In FIG. 12, the results are depicted. The signal increases linearly asthe channel depth increases. A straight regression line could becalculated (equation 1)

F(x)=6.2468x+50.016  (Equation 1)

Using the regression equation obtained (equation 1), the layerthicknesses between DNA chip and detection surface can now be determinedby means of the background fluorescence signal.

This was analyzed by stacking two glass surfaces (chips) havingstructured marks on their upper sides (crosses, numbers, and data matrixin FIG. 14), to which could be focused. The chips were stacked in such away that the structured marks were oriented towards each other and wereonly separated by a thin liquid layer. A 200 nM solution of aCy3-labeled oligonucleotide in 2×SSC+0.2% SDS was used as liquid. Usingthe focusing device of the microscope, which was provided with a scale,the distance between the marks and therefore the layer thickness of theliquid film could directly be determined. The intensity of thebackground is 158 gray values with an exposure time of 0.75 s. Thethickness of the gap as measured using the fluorescence microscope, is40 μm. Assuming that the measured gray values behave linearly inrelation to exposure time (see FIG. 13), according to equation 1 theresulting thickness of the gap is 42.6 μm. The values for the thicknessof the liquid layer thus obtained are well in agreement with each other.

b) Experiments for Reducing or Preventing Background Fluorescence byMeans of Compressing the Process Unit

In these experiments, the hybridization signal was measured depending onthe displacement of the fluorescent analyte caused by applying pressurevia a tappet. The experimental setup is shown in FIG. 15. By applyingpressure via the tappet, the silicon chip (3.15×3.15 mm) was pressedtowards a probe chip (DNA chip), and in this process the liquid locatedbetween the two surfaces was displaced.

For performing the experiment, the chamber was filled with ahybridization solution, representing a model system for the conditionsin a PCR hybridization. The hybridization solution included aCy3-labeled oligonucleotide (final concentration 2 nM in 2×SSC+0.2%SDS), which displayed complementarity to the probe array. In addition,the hybridization solution included another Cy3-labeled oligonucleotide,which does not hybridize with the probe array and therefore onlycontributes to the fluorescence background signal in the solution, butnot the specific signals at the spots.

Hybridization was performed for 10 min. For the subsequent reading outthe hybridization signals, a fixed exposure time of 1.5 s was selected.At the experimental setup, the tappet was pushed nearer towards theprobe array (detection surface) after each recording, so that the gapbetween array and second surface, which is filled with hybridizationsolution, was reduced.

FIG. 16 shows a recording of the hybridization signal with a thicknessof the gap of 10 μm. The results for background signal and hybridizationsignal at the spots are depicted in FIG. 17. As expected, both signalsbehave linearly in relation to the thickness of the gap. Thus, the spotsignal that is corrected by the background does not change with thethickness of the gap.

When a gray value of 255 is reached, the instrument is overloaded. Thatis., with a thickness of the gap of about 17 μm, measuring the spotintensity is only possible by reducing exposure time. For that reason,measuring sensitivity is then reduced.

Thus, the dynamic measuring range is increased by reducing the thicknessof the gap. By means of background adjustment of the spot signals(difference formation), the thickness of the gap can be varied in abroad range without influencing the measurement and the results. Withvery large thicknesses of the gap (>20 μm), measurement is stronglyimpaired due to overload of the detector.

c) Amplification, Hybridization and Detection as One-Stage Reaction

Two process units having a structure according to FIG. 15 were mountedand numbered.

Two identical reaction setups having the following composition wereprepared:

Reaction Setup:

20 mM dNTPs 0.5 μl 1 M potassium acetate (Kaac) 3 μl25 mM Mg-acetate Eppendorf 5 μl Clontech C-DNA PCR buffer 5 μlEppendorf Taq-polymerase 3 μl 10 μM primer CMV_DP_Cy3 1 μlCy3_5′TGAGGCTGGGAARCTGACA3′ 10 μM Primer CMV_UP_NH2 0.66 μl5′GGGYGAGGAYAACGAAATC3′_NH2 10 μM primer CMV_UP 0.33 μl5′GGGYGAGGAYAACGAAATC3′ 10 μM primer Entero_DP_Cy3 1 μlCy3_5′CCCTGAATGCGGCTAAT3′ 10 μM primer Entero_UP_NH2 0.66 μl5′ATTGTCACCATAAGCAGCC3′_NH2 lO μM primer Entero_UP 0.33 μl5′ATTGTCACCATAAGCAGCC3′ 10 μM primer HSV1_DP_Cy3 1 μlCy3_5′CTCGTAAAATGGCCCCTCC3′ 10 μM primer HSV1_UP_NH2 0.66 μl5′CGGCCGTGTGACACTATCG3′_NH2 10 μM primer HSV1_UP 0.33 μl5′CGGCCGTGTGACACTATCG 10 μM primer HSV2_UP_Cy3 1 μlCy3_5′CGCTCTCGTAAATGCT TCCCT3′ 10 μM primer HSV2_DP_NH2 0.66 μl5′TCTACCCACAACAGACCCAC G3′_NH2 10 μM primer HSV2_DP 0.33 μl5′TCTACCCACAACAGACCCACG3′ 10 μM primer VZV_DP_Cy3 1 μlCy3_5′TCGCGTGCTGCGGC 10 μM primer VZV_UP_NH2 0.66 μl5′CGGCATGGCCCGTCTAT3′_NH2 10 μM primer VZV_UP 0.33 μl5′CGGCATGGCCCGTCTAT Template CMV 1 μl PCR grade water 22.5 μl total 50μl

The process units were filled with 50 μl reaction setup each andprocessed according to the following temperature-time scheme.

1 Denaturation 95° C. Duration  300 s 2 Denaturation 95° C. Duration  10s 3 Annealing/Extension 60° C. Duration  20 s Repeating steps 2 to 3 35times 4 Denaturation 95° C. Duration  300 s 5 Hybridization 40° C.Duration 3600 s

Then, the two process units were subjected to different treatments. Inthe first case (process unit 1), the background fluorescence was reducedby displacing the analyte. This was accomplished by pushing the tappetupwards in the direction of the detection surface, so that the gapfilled with reaction solution is reduced as far as possible.

In the second case (process unit 2), the analyte was replaced by anon-fluorescent solution. The replacement of the solution was performedwith 2×SSC buffer at a fluctuation rate of 300 μl/min and a rinsingvolume of 900 μl. This procedure corresponds to the state of the art.

Subsequently, both strategies for reducing background fluorescence werecompared. To this end, the hybridization signals in both process unitswere detected with the aid of the fluorescence microscope camera setupdescribed.

Exposure time was 5 s (see FIG. 18 and FIG. 19). Comparing the spotintensities was performed on the basis of the spot comprising substanceCMV_S_21-3 (5′-NH2TGTTGGGCAACCACCGCACTG-3′). The location of the probesis indicated in FIGS. 18 and 19.

In FIG. 20, the result of the experiment is shown. By rinsing thereaction chamber in process unit 2, the hybridization signal is reducedcompared to the displacement in process unit 1. It is assumed that“bleeding” of the probes is responsible for this.

Thus, the method of analyte displacement according to the inventivemethod is to be preferred compared to replacement of the solutions.

In order to obtain evidence on amount and integrity of the amplificationproduct, 5 μl of each reaction solution were additionally analyzed on a2% agarose gel. The result (ethidium bromide-stained gel detected withan UV transilluminator) is shown in FIG. 21.

Example 4: Device for the Processing and Detection of Inventive ReactionCartridges

A device for the processing and detection of inventive reactioncartridges in accordance with this Example is shown in FIG. 28.

The device for performing microarray-based tests with reactioncartridges according to the present invention usually consists ofseveral components, which may be combined in one device or assembledmodularly from several partial devices. Optionally, the device may beactivated via an integrated computer or via an interface to an externalcomputer. The setup of the device is illustrated in FIG. 28.

An exemplary procedure is as follows:

The fluid interface of the reaction cartridge is manually brought in thefilling position by the operator, in which the cannulas penetrate theseal of the chamber body. Subsequently, the operator introduces thereaction mixture into the reaction chamber by means of a standardlaboratory pipette. Both steps can also be achieved by a correspondinglyconfigured device. The fluid interface is then again brought in the homeposition, wherein said procedure can also be achieved by acorrespondingly configured device.

The reaction cartridge is then inserted into the device. A data matrixreader, which is arranged in the device, recognizes the unique datamatrix attached to the reaction cartridge and, via a user-transmitteddata set, transfers the characteristic data for the cartridge as well asfor the test to be conducted to the control computer. This computer thencontrols the individual process steps, which can, for example, comprisean amplification and hybridization. Via the integrated pressure means,the capillary gap in the reaction chamber is subsequently reducedaccording to the present invention to allow for detection.

Detection can be performed with conventional fluorescence-opticalimaging or non-imaging systems. The data thus obtained are transmittedto a control computer for evaluation as well as presentation or storageon an internal or external interface.

Then, the reaction cartridge can be removed from the device anddiscarded by the operator.

Example 5: Reaction Cartridge Made of Electrically Conductive SyntheticMaterial

A reaction cartridge as depicted in FIG. 29 is prepared.

The lower shell (1) of the reaction cartridge consists of electricallyconductive synthetic material forming the base of the reaction chamber(Conduct 2, RKT, Germany). A foil PT-100 temperature sensor is fixed tothe bottom side of the chamber base using a suitable adhesive, forexample Loctite 401 (Loctite, Germany). Together with the seal (3) andthe coverslip (4), the lower shell forms the reaction chamber of thecartridge according to the present invention.

The cartridge further has a threaded drill hole (2) for inserting screwsfor electrical contacting, an upper shell (5) of the reaction cartridge,for example one made of acryl, a drill hole (6) for attaching the uppershell, and a detection window (7) within the upper shell.

A standard PCR reaction mixture is prepared:

30.5 μl de-ionized water 5 μl 10× PCR buffer (e.g., 10× cDNA PCRreaction buffer, Clontech, Germany) 5 μl Mg-acetate, 25 mM (e.g.,Eppendorf, Germany) 0.5 μl dNTP, 20 mM each 1 μl16sfD1 (5′-AGAGTTTGATCCTGGC TCAG-3′), 10 mM 1 μl16sRa (5′-TACCGTCACCATAAGGCTT CGTCCCTA-3′), 10 mM 3 μlTaq DNA polymerase (e.g., Genaxxon, Germany) 1 μl template

By using an insulin syringe (Becton Dickinson, Germany), the reactionchamber is filled with the reaction mixture. For ventilation during thefilling procedure, a second cannula is penetrated through the seal ofthe chamber body. After filling, ventilation cannula and insulin syringeare properly discarded.

The chamber is then connected to a regulating unit (CLONDIAG chiptechnologies GmbH, Germany) via the two screws provided for thispurpose. Likewise, the temperature sensor is connected to saidregulating unit at the bottom side of the lower shell. Said regulatingunit is capable of regulating specific temperatures in the lower shellaccording to a predefined program.

In this manner, the following PCR program is conducted: 5 min 95° C.,30× (30 s 95° C., 30 s 62° C., 50 s 72° C.).

FIG. 30 shows an image of the reaction cartridge recorded using athermal imaging camera at a temperature of 95° C.

After completion of the program, the reaction product is removed fromthe reaction chamber by means of an insulin syringe. Analogously, acannula is penetrated through the seal of the chamber body forventilation during the emptying of the reaction chamber.

The reaction product is analyzed by agarose gel electrophoresis. To thisend, 5 μl of the reaction solution, along with a suitable buffer (forexample 5 μl 250 mM in 50% glycerin, bromphenol blue), are applied tothe pocket of a 2% agarose gel and an electrophoresis is performed. Theresult is depicted in FIG. 31.

As can clearly be seen, an amplification product of correct size and inan amount comparable to the positive control could be obtained in allcases.

Example 6: Reaction Cartridge Having a Displacement Structure on theSecond Surface

A reaction cartridge as shown in FIG. 5 or 6 is prepared. In the centerof the second surface of the inventive device a drop (about 20 μl) ofSylgard 184 is deposited using a pin. Subsequently, the second surfacewith the silicone drop is incubated, for example, in an oven at 120° C.for one hour in order to cross-link the Sylgard. Subsequently saidsecond surface is assembled in a device according to the presentinvention. A DNA probe array is applied onto the first surface.

A PCR setup is prepared according to the following scheme:

10 x Clontech cDNA buffer  20 μl 25 mM Mg acetate Eppendorf  20 μldNTP's 20 mM each  2 μl Genaxxon Taq polymerase  12 μl Bidest 122 μlPrimer 1 10 μM with Cy3 label  4 μl Primer 2 10 μM  4 μl 1M K acetate 12 μl Template DNA  4 μl

20 μl of this mixture are introduced in the inventive device.Subsequently, the device is connected to a corresponding controller(prototype, Join, Jena, Germany), and a PCR is performed according tothe following scheme.

1) Denaturation 95° C. 500 s 2) Denaturation 95° C.  10 s 3) Annealing60° C.  30 s 4) Elongation 72° C.  30 s 5) 37 times repeating steps 2 to4 6) Denaturation 95° C.  60 s 7) Hybridization 50° C. 45 min

Then, the first surface is guided towards the second surface until thedisplacement structure contacts the surface area of the first surface.By flattening the elastic displacement structure on the surface of themicroarray, the fluorescent solution causing the background is displacedcompletely and the signal can be detected. (see FIG. 33).

Example 7: Detection of CD4 Antigens on Lymphocytes

As was mentioned above, the methods according to the present inventioncan also be performed by means of devices, wherein the reaction chamberformed by a first and a second surface is formed by different objects.As an example, it was referred to a reaction vessel, into which asnug-fit tappet, piston, or stamp is inserted in order to form acorrespondingly narrow reaction chamber from which the analyte liquid isdisplaced. This principle has been explained by way of FIG. 34.

Concretely, a micro well plate made of polystyrene having anamino-modified surface (for example nunc Immobilizer F96 Micro WellPlate—Amino)) is coated with anti-CD4 antibody (Molecular Probes). Theantibody is incubated at a concentration of 0.2 μg/μl in 1×PBS buffer inthe plate at 23° C. for 1 h and the plate is subsequently washed with1×PBS buffer. 40 μl of a blood sample are mixed with 160 μl deionizedwater, 40 μl 0.5 M EDTA solution and 5 μl of a solution of an anti-CD4antibody labeled with the fluorescence dye phycoerythrin in 1×PBS(Molecular Probes, original concentration 0.2 mg/ml), are briefly shakenand transferred to a well in the plate coated with capture-antibody.

After short incubation (15 min) in a shaker (500 r/min) at roomtemperature, the plate is read out on an inverse fluorescencemicroscope. To this end, tappets are inserted into the wells of theplate, which come to rest directly on the surface of the plate and havethe characteristic of substantially displacing the liquid and notemitting fluorescence light within the selected detection range(excitation: 541 nm, emission:576 nm) themselves. The signal is recordedby means of a CCD camera and compared to the signal measured in otherwells of the plate.

Example 8: Detection of the Number of Lymphocytes Bearing CD4 Antigen

As was mentioned above, the methods according to the present inventioncan also be performed if the probes are not immobilized to the firstsurface of the reaction chamber. This aspect of the invention wasexplained by way of FIGS. 35 and 36.

Concretely, 1 μl of a blood sample is mixed with 4 μl deionized water, 1μl 0.5 M EDTA solution and 0.5 μl of a solution of an anti-CD4 antibodylabeled with the fluorescence dye phycoerythrin in 1×PBS (MolecularProbes, original concentration 0.2 mg/ml), briefly shaken andtransferred to a compressible detection chamber.

The exemplary compressible detection chamber is generated by means of acartridge as it was described above and is, for example, depicted in theFIGS. 3 to 6 or in the FIGS. 10 and 11, but without using the chipequipped with the heating sensor, however.

Instead, a PDMS membrane having a thickness of about 0.3 μm is used asfirst surface. For detection, said membrane is pressed against thedetection plane and read out by means of a fluorescence microscope(Axioskop, Zeiss, Germany, excitation: 541 nm, emission:576 nm).

The result is depicted in FIG. 37. The dark spots (a), (exemplary) areerythrocytes, which are not recognized by the anti-CD4 antibody. Thelight spots (b) are lymphocytes. In the present experiment, 5lymphocytes can thus be detected in the detection volume.

If a substrate is mentioned in the above-mentioned Figures, this is tobe understood to denote a substrate, in which probes can be immobilized,such as, for example, a microarray. In case there are no probesimmobilized, the term substrate is, in this context, supposed to definethe region of the first surface or the second surface, where thedetection of the targets is supposed to take place in the state of thecompressed reaction chamber. In both cases (i.e. immobilized probe andnon-immobilized probe, respectively), the substrate does not have to bean individual component, but it can represent a region of the firstsurface (immobilized probes) or of the first and/or second surface(non-immobilized probes).

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the invention. All references identified hereinare incorporated by reference in their entirety. Accordingly, otherembodiments are within the scope of the following claims.

1-62. (canceled)
 63. A device for qualitatively and/or quantitativelydetecting molecular interactions between probe molecules and targetmolecules in a sample solution, said device comprising: a micro-arrayhaving a substrate, on which probe molecules are immobilized on arrayelements, said micro-array being arranged on a first surface of thedevice; and a reaction chamber formed between the first surface havingsaid micro-array arranged thereon, and a second surface, wherein thedistance between said micro-array and said second surface is variablesuch that the sample solution between said micro-array and said secondsurface can be essentially removed without removing the sample solutionfrom the reaction chamber.
 64. The device according to claim 63, whereinthe device further comprises a means for varying the distance betweensaid micro-array and said second surface in such a way that the samplesolution between said micro-array and said second surface can beessentially removed.
 65. The device according to claim 64, wherein thedistance between said micro-array and the second surface is variable ina range of about 0 to about 1 mm.
 66. The device according to claim 63,wherein the device further comprises a temperature control unit and/ortemperature regulating unit for controlling and/or regulating thetemperature within the reaction chamber.
 67. The device according toclaim 63, wherein the device comprises a detection system and afluorescence-optical system.
 68. The device according to claim 67,wherein said fluorescence-optical system is a fluorescence microscopewithout an autofocus.
 69. The device according to claim 67, wherein saiddetection system is connected to a spacer which, when resting upon thesecond surface, adjusts a spacing between the detection system and thesecond surface.
 70. The device according to claim 67, wherein laterallylimiting compensation zones are provided for the reaction chamber formedbetween the first and second surfaces, said compensation zones keepingthe volume within the reaction chamber upon a reduction of the spacingbetween said micro-array and the second surface essentially constant.71. The device according claim 63, wherein the second surface is made ofan optically transparent material.
 72. The device according to claim 63,wherein the first surface is configured at least in the zone below saidmicro-array such that said micro-array may be guided relative to thesecond surface so that the distance between said micro-array and thesecond surface is variable.
 73. The device according to claim 72,wherein the first surface is elastically deformable at least in the zonebelow said micro-array, and wherein the first surface is made of elasticplastics.
 74. The device according to claim 63, wherein said micro-arraymay be guided relative to the second surface by said means for varyingthe distance between said micro-array and said second surface, andwherein said micro-array is guided relative to the second surface bysaid means acting upon the first surface by compression and/or tensionand/or wherein the first surface is set into vibration by said means.75. The device according to claim 63, wherein the second surface may beguided relative to the first surface so that the distance between saidmicro-array and the second surface is variable, wherein the secondsurface is guided relative to the first surface by said spacer actingupon the second surface by compression and/or tension so that thedistance between said micro-array and the second surface is variable.76. The device according to claim 63, wherein the probe molecules and/ortarget molecules are biopolymers selected from the group consisting ofnucleic acids, peptides, proteins, antigens, antibodies, carbohydratesand/or analogs thereof, and/or copolymers of the above-mentionedbiopolymers.